Apparatus and methods for biosynthetic production of cannabinoids

ABSTRACT

The present invention provides an apparatus and methods for producing tetrahydrocannabinolic acid (THCA), cannabichromenic acid (CBCA) and cannabichromenic acid (CBCA) in different ratios. The apparatus comprises: (i) a bioreactor comprising (a) an automated supply system configured to deliver a first automated supply of cannabigerolic acid (CBGA), a cannabinoid acid synthase, and a reaction mixture; and (b) a second automated system to cease the reaction; (ii) a controller configured to modify a property of the reaction mixture to produce the desired products; and (iii) an extractor configured to recover the tetrahydrocannabinolic acid (THCA), cannabichromenic acid (CBCA) or cannabidiolic acid (CBDA) and cannabichromenic acid.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of U.S. application Ser. No.15/232,405 filed Aug. 9, 2016, incorporated herein by reference in itsentirety, which is a Continuation of U.S. application Ser. No.15/158,565, filed May 18, 2016, now U.S. Pat. No. 9,512,391 issued Dec.6, 2016, incorporated herein by reference in its entirety, which is aContinuation of U.S. application Ser. No. 14/835,444, filed Aug. 25,2015, now U.S. Pat. No. 9,394,510 issued Jul. 19, 2016, incorporatedherein by reference in its entirety, which claims priority fromProvisional U.S. Application 62/041,521, filed Aug. 25, 2014,incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to the biosynthesis of cannabinoids.Specifically, the present invention relates to the production andmanipulation of enzymes involved in the synthesis of cannabinoids, andto the simultaneous synthesis of various cannabinoids in differentratios.

BACKGROUND OF THE INVENTION

Cannabinoids are terpenophenolic compounds found in Cannabis sativa, anannual plant belonging to the Cannabaceae family. The plant containsmore than 400 chemicals and approximately 70 cannabinoids. The latteraccumulate mainly in the glandular trichomes. The most active of thenaturally occurring cannabinoids is tetrahydrocannabinol (THC), which isused for treating a wide range of medical conditions, includingglaucoma, AIDS wasting, neuropathic pain, treatment of spasticityassociated with multiple sclerosis, fibromyalgia andchemotherapy-induced nausea. THC is also effective in the treatment ofallergies, inflammation, infection, epilepsy, depression, migraine,bipolar disorders, anxiety disorder, drug dependency and drug withdrawalsyndromes.

Additional active cannabinoids include cannabidiol (CBD), an isomer ofTHC, which is a potent antioxidant and anti-inflammatory compound knownto provide protection against acute and chronic neuro-degeneration;cannabigerol (CBG), found in high concentrations in hemp, which acts asa high affinity α₂-adrenergic receptor agonist, moderate affinity5-HT_(1A) receptor antagonist and low affinity CB1 receptor antagonist,and possibly has anti-depressant activity; and cannabichromene (CBC),which possesses anti-inflammatory, anti-fungal and anti-viralproperties. Many phytocannabinoids have therapeutic potential in avariety of diseases and may play a relevant role in plant defense aswell as in pharmacology. Accordingly, biotechnological production ofcannabinoids and cannabinoid-like compounds with therapeutic propertiesis of uttermost importance. Thus, cannabinoids are considered to bepromising agents for their beneficial effects in the treatment ofvarious diseases.

Despite their known beneficial effects, therapeutic use of cannabinoidsis hampered by the high costs associated with the growing andmaintenance of the plants in large scale and the difficulty in obtaininghigh yields of cannabinoids. Extraction, isolation and purification ofcannabinoids from plant tissue is particularly challenging ascannabinoids oxidize easily and are sensitive to light and heat. Inaddition, although it has been hypothesized that CBCA is predominantlysynthesized from CBGA by the enzyme CBCA synthase, the enzyme has notbeen isolated or cloned. There is therefore a need for developingmethodologies that allow large-scale production of cannabinoids fortherapeutic use. The present invention addresses this need.

SUMMARY OF THE INVENTION

It is therefore an object of the invention to provide solutions to theaforementioned deficiencies in the art. To this end the inventionprovides a method of producing one or more cannabinoids or cannabinoidanalogs comprising the steps of: (a) selecting a compound according toFormula I:

(b) selecting a cannabinoid acid synthase as a catalyst for transformingthe compound according to Formula I into one or more cannabinoids orcannabinoid analogs; (c) reacting the compound of Formula I with thecannabinoid acid synthase in a reaction mixture comprising a solvent andan amphiphilic compound; (d) isolating from the reaction mixture one ormore cannabinoid acids or cannabinoid analogs produced in step (c); and(e) optionally decarboxylating the cannabinoid acids or cannabinoidanalogs isolated in step (c); wherein R is selected from —OH, halogen,—SH, or a —NR_(a)R_(b) group; R₁ and R₂ are each independently selectedfrom the group consisting of —H, —C(O)R_(a), —OR_(a), an optionallysubstituted C₁-C₁₀ linear or branched alkylene, an optionallysubstituted C₂-C₁₀ linear or branched alkenylene, an optionallysubstituted C₂-C₁₀ linear or branched alkynylene, an optionallysubstituted C₃-C₁₀ aryl, an optionally substituted C₃-C₁₀ cycloalkyl,(C₃-C₁₀)aryl-(C₁-C₁₀)alkylene, (C₃-C₁₀)aryl-(C₂-C₁₀)alkenylene, and(C₃-C₁₀)aryl-(C₁-C₁₀)alkynylene, or R₁ and R₂ together with the carbonatoms to which they are bonded form a C₅-C₁₀ cyclic ring; R₃ is selectedfrom the group consisting of H, —C(O)R_(a) and C₁-C₁₀ linear or branchedalkyl; and R_(a) and R_(b) are each independently —H, —OH, —SH, —NH₂,(C₁-C₁₀) linear or branched alkyl, or a C₃-C₁₀ cycloalkyl.

Preferably, the cannabinoid acid synthase is cannabidiolic acid (CBDA)synthase or tetrahydrocannabinolic acid (THCA) synthase. In a preferredaspect of the invention, the C₅-C₁₀ cyclic ring comprises one or moreheteroatoms selected from oxygen, sulfur or nitrogen. In anotherpreferred aspect of the invention, R₂ is a linear alkylene selected fromthe group consisting of CH₃, C₂H₅, C₃H₇, C₄H₉, C₅H₁₁, C₆H₁₃, C₇H₁₅ andC₈H₁₇. Preferably, R₂ is a C₂-C₁₀ alkenylene selected from the groupconsisting of

and R₄ is a linear alkylene selected from the group consisting of CH₃,C₂H₅, C₃H₇, C₄H₉, C₅H₁₁, C₆H₁₃, C₇H₁₅ and C₈H₁₇. In another preferredaspect, R₂ is a C₂-C₁₀ linear or branched alkynylene selected from thegroup consisting of

In an additional preferred embodiment, R₂ is

wherein X is —OH, —SH, or —NR_(a)R_(b), and wherein R_(a) and R_(b) areeach independently —H, —OH, —SH, —NH₂, (C₁-C₁₀) linear or branchedalkyl, or a C₃-C₁₀ cycloalkyl. Most preferably, R is —OH, R₁ is —COOH,R₂ is C₅H₁₁ and R₃ is —H.

In one embodiment, the cannabinoid acid synthase is a recombinantcannabinoid acid synthase obtained by generating one or more copies of acannabinoid acid synthase gene and overexpressing a protein encoded bythe cannabinoid acid synthase gene. In a preferred aspect of theinvention, one or more copies of a cannabinoid acid synthase gene aregenerated in vivo and the method comprises step (i) of integrating oneor more copies of the cannabinoid acid synthase gene into the genome ofan eukaryotic host to scale up protein expression. Preferably, theeukaryotic host is Pichia pastoris and the cannabinoid acid synthasegene is codon optimized with an alpha secretion sequence to targetprotein secretion and tagged with six tandem histidine residues (SEQ IDNO: 9). Step (i) may comprise linearizing the cannabinoid acid synthasegene by digestion with one or more restriction enzymes; extracting thecannabinoid acid synthase gene by gel extraction; ligating thecannabinoid acid synthase gene into a Pichia pastoris plasmid; andelectroporating the plasmid into bacterial cells to generate one or morecannabinoid acid synthase gene copy colonies.

In a preferred aspect of the invention, the solvent is DMSO, and theconcentration of DMSO in the reaction mixture is 20% (w/v). In anadditional preferred aspect, the amphiphilic compound is a surfactant ora cyclodextrin. In a preferred embodiment, the cyclodextrin isα-cyclodextrin, β-cyclodextrin or γ-cyclodextrin. Even more preferably,the cyclodextrin is sulfobuthylether β-cyclodextrin sodium salt orrandomly methylated β-cyclodextrin, and the concentration ofcyclodextrin in the reaction mixture is between 2 and 28 mg/ml. In amost preferred embodiment, the concentration of cyclodextrin in thereaction mixture is 8 mg/ml.

In one embodiment, the cannabinoids or cannabinoid analogs are singleenantiomers with an enantiomeric purity of at least 95%, and preferablyof at least 99%.

In a preferred embodiment, the cannabinoid acid synthase is THCAsynthase and the one or more cannabinoids or cannabinoid analogs aretetrahydrocannabinol (THCA), cannabichromene (CBCA), THCA and CBCA, oranalogs thereof. In a preferred aspect, the amphiphilic compound iscyclodextrin and the mass:mass ratio of cyclodextrin to the compound ofFormula I is 28:1 or the molar ratio of cyclodextrin to the compound ofFormula I is 7.3:1. Preferably, step (c) of the reaction is performed ata pH in a range between 3.8 and 7.2, and the method produces THCA, CBCA,or THCA and CBCA in a ratio as shown in the following table at eachspecified pH:

pH THCA CBCA 4 1 0 5 2.33 1 6 1 5.67 7 0 1 8 0 0

Preferably, 98% of the compound of Formula I is converted into one ormore cannabinoids or cannabinoid analogs within two hours.

In a different embodiment, the cannabinoid acid synthase is CBDAsynthase and the method produces cannabidiol (CBDA), cannabichromeneacid (CBCA), CBDA and CBCA, or analogs thereof. Preferably, theamphiphilic compound is cyclodextrin and the mass:mass ratio ofcyclodextrin to the compound of Formula I is 11:1 or the molar ratio ofcyclodextrin to the compound of Formula I is 4:1. Preferably, step (c)is performed at a pH in a range between 3.8 and 7.2. In a preferredembodiment, the method produces CBDA, CBCA, or CBDA and CBCA in a ratioas shown in the following table at each specified pH:

pH CBDA CBCA 4.2 2.5 1 5 1.13 1 5.2 1 1.17 5.4 1 2.45 5.8 1 6.14 6.2 128.13 6.8 0 0

Most preferably, 98% of the compound of Formula I is converted into oneor more cannabinoids or cannabinoid analogs within two hours.

In a different embodiment, the invention provides a method of producingone or more cannabinoids or cannabinoid analogs according to Formula II

wherein the method comprises the steps of: (a) reacting a compoundaccording to Formula III with a compound according to Formula IV;

in the presence of an enzyme that catalyzes the reaction of the FormulaIII and Formula IV compounds to form a Formula II compound; (b) reactingthe Formula II compound with a cannabinoid acid synthase in a reactionmixture comprising a solvent and an amphiphilic compound to produce oneor more cannabinoids or cannabinoid analogs; (c) isolating from thereaction mixture one or more cannabinoids or cannabinoid analogsproduced in step (b); and (e) optionally decarboxylating the one or morecannabinoids or cannabinoid analogs isolated in step (c); wherein R isselected from —OH, halogen, —SH, or a —NR_(a)R_(b) group; R₁ and R₂ areeach independently selected from the group consisting of —H, —C(O)R_(a),—OR_(a), an optionally substituted linear or branched (C₁-C₁₀)alkylene,an optionally substituted linear or branched (C₂-C₁₀)alkenylene, anoptionally substituted linear or branched (C₂-C₁₀)alkynylene, anoptionally substituted C₃-C₁₀ aryl, an optionally substituted C₃-C₁₀cycloalkyl, (C₃-C₁₀)aryl-(C₁-C₁₀)alkylene,(C₃-C₁₀)aryl-(C₂-C₁₀)alkenylene, and (C₃-C₁₀)aryl-(C₁-C₁₀)alkynylene, orR₁ and R₂ together with the carbon atoms to which they are bonded form aC₅-C₁₀ cyclic ring; R₃ is selected from the group consisting of H,—C(O)R_(a) and C₁-C₁₀ linear or branched alkyl; R₅ is selected from thegroup consisting of a linear or branched (C₁-C₁₀)alkylene, a linear orbranched (C₂-C₁₀)alkenylene, a linear or branched (C₂-C₁₀)alkynylene,—C(O)—(C₁-C₁₀)alkylene, —C(O)—(C₂-C₁₀)alkenylene and—C(O)—(C₂-C₁₀)alkynylene; wherein any alkylene, alkenylene, alkynylene,aryl, arylalkylene, or cycloalkyl group is further substituted with oneor more groups selected from the group consisting of —OH, halogen,—NR_(b)R_(c), —C(O)R_(a), —C(O)NR_(b)R_(c), (C₁-C₁₀)alkyl, —CN,(C₁-C₄)alkoxy, (C₁-C₄)haloalkyl, and (C₁-C₄)hydroxyalkyl; and R_(a),R_(b) and R_(c), are each independently —H, —OH, —SH, —NH₂, (C₁-C₁₀)linear or branched alkyl, or a C₃-C₁₀ cycloalkyl.

In one embodiment, R₅ is a (C₂-C₁₀)alkenylene selected from the groupconsisting of

and R₄ is a linear alkylene selected from the group consisting of CH₃,C₂H₅, C₃H₇, C₄H₉, C₅H₁₁, C₆H₁₃, C₇H₁₅ and C₈H₁₇. In a preferred aspectof the invention, R₅ is

and R₆ is selected from (C₁-C₁₀)alkylene, (C₂-C₁₀)alkenylene, —OH, —SH,NO₂, F, Cl, Br, —NH₂, or —NHR_(a).

In another preferred embodiment, the cannabinoid acid synthase is arecombinant cannabinoid acid synthase obtained by generating one or morecopies of a cannabinoid acid synthase gene and by overexpressing aprotein encoded by the cannabinoid acid synthase gene. Preferably, oneor more copies of a cannabinoid acid synthase gene are generated in vivoand the method comprises step (i) of integrating one or more copies ofthe cannabinoid acid synthase gene into the genome of a eukaryotic hostto scale up protein expression. Preferably, the eukaryotic host isPichia pastoris and the cannabinoid acid synthase gene is codonoptimized with an alpha secretion sequence to target protein secretionand tagged with six tandem histidine residues (SEQ ID NO: 9). Step (i)may comprise linearizing the cannabinoid acid synthase gene by digestionwith one or more restriction enzymes; extracting the cannabinoid acidsynthase gene by gel extraction; ligating the cannabinoid acid synthasegene into a Pichia pastoris plasmid; and electroporating the plasmidinto bacterial cells to generate one or more cannabinoid acid synthasegene copy colonies.

In a preferred aspect of the invention, the solvent is DMSO, and theconcentration of DMSO in the reaction mixture is 20% (w/v). In anadditional preferred aspect, the amphiphilic compound is a surfactant ora cyclodextrin. In a preferred embodiment, the cyclodextrin isα-cyclodextrin, β-cyclodextrin or γ-cyclodextrin. Even more preferably,the cyclodextrin is sulfobuthylether β-cyclodextrin sodium salt orrandomly methylated β-cyclodextrin, and the concentration ofcyclodextrin in the reaction mixture is between 2 and 28 mg/ml. In amost preferred embodiment, the concentration of cyclodextrin in thereaction mixture is 8 mg/ml.

In one embodiment, the cannabinoids or cannabinoid analogs are singleenantiomers with an enantiomeric purity of at least 95%, and preferablyof at least 99%.

In a preferred embodiment, the cannabinoid acid synthase is THCAsynthase and the one or more cannabinoids or cannabinoid analogs aretetrahydrocannabinol (THCA), cannabichromene (CBCA), THCA and CBCA, oranalogs thereof. In a preferred aspect, the amphiphilic compound iscyclodextrin and the mass:mass ratio of cyclodextrin to the compound ofFormula I is 28:1 or the molar ratio of cyclodextrin to the compound ofFormula I is 7.3:1. Preferably, step (c) of the reaction is performed ata pH in a range between 3.8 and 7.2, and the method produces THCA, CBCA,or THCA and CBCA in different ratios as described above. Preferably, 98%of the compound of Formula I is converted into one or more cannabinoidsor cannabinoid analogs within two hours.

In a different embodiment, the cannabinoid acid synthase is CBDAsynthase and the method produces cannabidiol (CBDA), cannabichromeneacid (CBCA), CBDA and CBCA, or analogs thereof. Preferably, theamphiphilic compound is cyclodextrin and the mass:mass ratio ofcyclodextrin to the compound of Formula I is 11:1 or the molar ratio ofcyclodextrin to the compound of Formula I is 4:1. Preferably, step (c)is performed at a pH in a range between 3.8 and 7.2, and the methodproduces CBDA, CBCA, or CBDA and CBCA in different ratios as describedabove. Most preferably, 70% of the compound of Formula I is convertedinto one or more cannabinoids or cannabinoid analogs within two hours.

In yet another embodiment, the invention provides a method for producinga tetrahydrocannabinol, cannabichromene, or both tetrahydrocannabinoland cannabichromene, or their analogs, wherein the method comprises thesteps of: (a) selecting a compound according to Formula I;

(b) reacting the compound of Formula I with a tetrahydrocannabinolicacid (THCA) synthase in a reaction mixture comprising a solvent and anamphiphilic compound; (c) modifying at least one property of thereaction mixture to obtain a tetrahydrocannabinol, a cannabichromene, orboth tetrahydrocannabinol and cannabichromene, or their analogs asproducts; (d) isolating tetrahydrocannabinol, cannabichromene, or bothtetrahydrocannabinol and cannabichromene, or their analogs from thereaction mixture; and (e) decarboxylating the tetrahydrocannabinolicacid, cannabichromenic acid, or both tetrahydrocannabinolic acid andcannabichromenic acid, or their analogs; wherein R is selected from —OH,halogen, —SH, or a —NR_(a)R_(b) group; R₁ and R₂ are each independentlyselected from the group consisting of —H, —C(O)R_(a), —OR_(a), anoptionally substituted C₁-C₁₀ linear or branched alkylene, an optionallysubstituted C₂-C₁₀ linear or branched alkenylene, an optionallysubstituted C₂-C₁₀ linear or branched alkynylene, an optionallysubstituted C₃-C₁₀ aryl, an optionally substituted C₃-C₁₀ cycloalkyl,(C₃-C₁₀)aryl-(C₁-C₁₀)alkylene, (C₃-C₁₀)aryl-(C₂-C₁₀)alkenylene, and(C₃-C₁₀)aryl-(C₁-C₁₀)alkynylene, or R₁ and R₂ together with the carbonatoms to which they are bonded form a C₅-C₁₀ cyclic ring; R₃ is selectedfrom the group consisting of —H, —C(O)R_(a) and C₁-C₁₀ linear orbranched alkyl; and R_(a) and R_(b) are each independently —H, —OH, —SH,—NH₂, (C₁-C₁₀) linear or branched alkyl, or a C₃-C₁₀ cycloalkyl.

In a preferred embodiment, the THCA synthase is a recombinant THCAsynthase obtained by generating one or more copies of a THCA synthasegene and by overexpressing a protein encoded by the THCA synthase gene.Preferably, one or more copies of the THCA synthase gene are generatedin vivo and the method comprises step (i) of integrating one or morecopies of the cannabinoid acid synthase gene into the genome of aeukaryotic host to scale up protein expression. Preferably, theeukaryotic host is Pichia pastoris and the THCA synthase gene is codonoptimized with an alpha secretion sequence to target protein secretionand tagged with six tandem histidine residues (SEQ ID NO: 9). Step (i)may comprise linearizing the cannabinoid acid synthase gene by digestionwith one or more restriction enzymes; extracting the cannabinoid acidsynthase gene by gel extraction; ligating the cannabinoid acid synthasegene into a Pichia pastoris plasmid; and electroporating the plasmidinto bacterial cells to generate one or more cannabinoid acid synthasegene copy colonies.

In a preferred aspect of the invention, the solvent is DMSO, and theconcentration of DMSO in the reaction mixture is 20% (w/v). In anadditional preferred aspect, the amphiphilic compound is a surfactant ora cyclodextrin. In a preferred embodiment, the cyclodextrin isα-cyclodextrin, β-cyclodextrin or γ-cyclodextrin. Even more preferably,the cyclodextrin is sulfobuthylether β-cyclodextrin sodium salt orrandomly methylated β-cyclodextrin, and the concentration ofcyclodextrin in the reaction mixture is between 2 and 28 mg/ml. In amost preferred embodiment, the concentration of cyclodextrin in thereaction mixture is 8 mg/ml.

In a preferred aspect of the invention, the amphiphilic compound iscyclodextrin and the mass:mass ratio of cyclodextrin to the compound ofFormula I is 28:1 or the molar ratio of cyclodextrin to the compound ofFormula I is 7.3:1. Preferably, step (c) of modifying at least oneproperty of the reaction mixture comprises modifying the pH of thereaction mixture in a range between 3.8 and 7.2, and the method producesTHCA, CBCA, or THCA and CBCA in different ratios as described above.Preferably, 98% of the compound of Formula I is converted into one ormore cannabinoids or cannabinoid analogs within two hours.

In a different embodiment, the invention provides a method for producinga cannabidiol, cannabichromene, or both cannabidiol and cannabichromene,or their analogs comprising the steps of: (a) selecting a compoundaccording to Formula I;

(b) reacting the compound of Formula I with a cannabinodiolic acid(CBDA) synthase in a reaction mixture comprising a solvent and anamphiphilic compound; (c) modifying at least one property of thereaction mixture to obtain a cannabidiol, a cannabichromene, or bothcannabidiol and cannabichromene, or their analogs as products; (d)isolating cannabidiol, cannabichromene, or both cannabidiol andcannabichromene, or their analogs from the reaction mixture; and (e)decarboxylating the cannabidiol, cannabichromene, or both cannabidioland cannabichromene, or their analogs; wherein R is selected from —OH,halogen, —SH, or a —NR_(a)R_(b) group; R₁ and R₂ are each independentlyselected from the group consisting of —H, —C(O)R_(a), —OR_(a), anoptionally substituted C₁-C₁₀ linear or branched alkylene, an optionallysubstituted C₂-C₁₀ linear or branched alkenylene, an optionallysubstituted C₂-C₁₀ linear or branched alkynylene, an optionallysubstituted C₃-C₁₀ aryl, an optionally substituted C₃-C₁₀ cycloalkyl,(C₃-C₁₀)aryl-(C₁-C₁₀)alkylene, (C₃-C₁₀)aryl-(C₂-C₁₀)alkenylene, and(C₃-C₁₀)aryl-(C₁-C₁₀)alkynylene, or R₁ and R₂ together with the carbonatoms to which they are bonded form a C₅-C₁₀ cyclic ring; R₃ is selectedfrom the group consisting of H, —C(O)R_(a) and C₁-C₁₀ linear or branchedalkyl; and R_(a) and R_(b) are each independently —H, —OH, —SH, —NH₂,(C₁-C₁₀) linear or branched alkyl, or a C₃-C₁₀ cycloalkyl.

Preferably, the CBDA synthase is a recombinant CBDA synthase obtained bygenerating one or more copies of a CBDA synthase gene and byoverexpressing a protein encoded by the CBDA synthase gene. In apreferred aspect of the invention, one or more copies of a CBDA synthasegene are generated in vivo and the method comprises step (i) ofintegrating one or more copies of the CBDA synthase gene into the genomeof a eukaryotic host to scale up protein expression. Preferably, theeukaryotic host is Pichia pastoris and the CBDA synthase gene is codonoptimized with an alpha secretion sequence to target protein secretionand tagged with six tandem histidine residues (SEQ ID NO: 9). Step (i)may comprise linearizing the CBDA synthase gene by digestion with one ormore restriction enzymes; extracting the CBDA synthase gene by gelextraction; ligating the CBDA synthase gene into a Pichia pastorisplasmid; and electroporating the plasmid into bacterial cells togenerate one or more cannabinoid acid synthase gene copy colonies.

In a preferred aspect of the invention, the solvent is DMSO, and theconcentration of DMSO in the reaction mixture is 20% (w/v). In anadditional preferred aspect, the amphiphilic compound is a surfactant ora cyclodextrin. In a preferred embodiment, the cyclodextrin isα-cyclodextrin, β-cyclodextrin or γ-cyclodextrin. Even more preferably,the cyclodextrin is sulfobuthylether β-cyclodextrin sodium salt orrandomly methylated β-cyclodextrin, and the concentration ofcyclodextrin in the reaction mixture is between 2 and 28 mg/ml. In amost preferred embodiment, the concentration of cyclodextrin in thereaction mixture is 8 mg/ml.

In a preferred aspect of the invention, the amphiphilic compound iscyclodextrin and the mass:mass ratio of cyclodextrin to the compound ofFormula I is 28:1(w/w) or the molar ratio of cyclodextrin to thecompound of Formula I is 7.3:1. Preferably, step (c) of modifying atleast one property of the reaction mixture comprises modifying the pH ofthe reaction mixture in a range between 3.8 and 7.2, and the methodproduces CBDA, CBCA, or CBDA and CBCA in different ratios as describedabove. In a preferred embodiment, 98% of the compound of Formula I isconverted into one or more cannabinoids or cannabinoid analogs withintwo hours.

In a different embodiment, the invention provides a system for producingone or more cannabinoids or cannabinoid analogs, comprising: a fermentorholding a medium and a plurality of cells, wherein the cells areconfigured to produce and secrete a cannabinoid synthase; a bioreactorcontaining a reactant in a reaction mixture comprising a solvent and anamphiphilic compound, the reactant configured to interact withcannabinoid acid synthase to form a first cannabinoid and a secondcannabinoid; and a control mechanism configured to control a conditionof the bioreactor, wherein the condition of the bioreactor influences aquantity formed of the first cannabinoid relative to a quantity formedof a second cannabinoid, and wherein the first and second cannabinoidsare each one a cannabinoid or a cannabinoid analog.

In a preferred embodiment, the bioreactor is a column bioreactorcontaining nickel, and the cannabinoid acid synthase includes a tagconfigured to bond to nickel. In some embodiments, the bioreactor is acolumn bioreactor containing both nickel and another metal.

In one embodiment, the reactant in the system is a compound according toFormula I;

Wherein R is selected from —OH, halogen, —SH, or a —NR_(a)R_(b) group;R₁ and R₂ are each independently selected from the group consisting of—H, —C(O)R_(a), —OR_(a), an optionally substituted C₁-C₁₀ linear orbranched alkylene, an optionally substituted C₂-C₁₀ linear or branchedalkenylene, an optionally substituted C₂-C₁₀ linear or branchedalkynylene, an optionally substituted C₃-C₁₀ aryl, an optionallysubstituted C₃-C₁₀ cycloalkyl, (C₃-C₁₀)aryl-(C₁-C₁₀)alkylene,(C₃-C₁₀)aryl-(C₂-C₁₀)alkenylene, and (C₃-C₁₀)aryl-(C₁-C₁₀)alkynylene, orR₁ and R₂ together with the carbon atoms to which they are bonded form aC₅-C₁₀ cyclic ring; R₃ is selected from the group consisting of H,—C(O)R_(a) and C₁-C₁₀ linear or branched alkyl; and R_(a) and R_(b) areeach independently —H, —OH, —SH, —NH₂, (C₁-C₁₀) linear or branchedalkyl, or a C₃-C₁₀ cycloalkyl.

In one preferred embodiment, the cannabinoid acid synthase iscannabidiolic acid (CBDA) synthase and the first and the secondcannabinoids are one or both of cannabidiolic acid and cannabichromenicacid or their analogs.

In another preferred embodiment, the cannabinoid acid synthase istetrahydrocannabinolic acid (THCA) synthase and the first and the secondcannabinoids are one or both of tetrahydrocannabinolic acid andcannabichromenic acid, or their analogs.

Preferably, the cannabinoid acid synthase interacts with the reactant inthe bioreactor to form both the first cannabinoid and the secondcannabinoid, and the condition of the bioreactor is a function of atleast one of pH, solvent, temperature, pressure, and flow rate.

In a preferred embodiment, a change in the condition of the bioreactoris configured to cause a shift from: 1) formation of the firstcannabinoid in greater quantities relative to the second cannabinoid to2) formation of the second cannabinoid in greater quantities relative tothe first cannabinoid.

Preferably the solvent in the system is DMSO, and the concentration ofDMSO in the reaction mixture is 20% (w/v).

In yet another preferred embodiment, the amphiphilic compound in thesystem is a surfactant or a cyclodextrin. Preferably, the cyclodextrinis α-cyclodextrin, β-cyclodextrin or γ-cyclodextrin. Even morepreferably, the cyclodextrin is sulfobuthylether β-cyclodextrin sodiumsalt or randomly methylated β-cyclodextrin, and the concentration ofcyclodextrin in the reaction mixture is between 2 and 28 mg/ml. Mostpreferably, the concentration of cyclodextrin in the reaction mixture is8 mg/ml.

In one aspect of the invention, the amphiphilic compound is cyclodextrinand the mass:mass ratio of cyclodextrin to the compound of Formula I is28:1 (w/w), or the molar ratio of cyclodextrin to the compound ofFormula I is 7.3:1. In a preferred embodiment, 98% of the compound ofFormula I in the system is converted into one or more cannabinoids orcannabinoid analogs within two hours.

In a preferred aspect of the invention, the cannabinoid acid synthase inthe system is CBDA synthase and the change in the condition of thebioreactor comprises modifying the pH of the reaction mixture in a rangebetween 3.8 and 7.2. Preferably, the method produces CBDA, CBCA, or CBDAand CBCA in different ratios as described above.

In another preferred embodiment, the cannabinoid acid synthase is THCAsynthase and the change in the condition of the bioreactor comprisesmodifying the pH of the reaction mixture in a range between 3.8 and 7.2.Preferably, the method produces THCA, CBCA, or THCA and CBCA indifferent ratios as described above.

In yet another embodiment, the invention provides a method for producingat least one cannabinoid or cannabinoid analog, that includes the stepsof: providing cannabigerol, a cannabinoid acid synthase, and a reactionmixture comprising a solvent and an amphiphilic compound via anautomated delivery system; reacting the cannabigerol with thecannabinoid acid synthase in the reaction mixture; adding a solvent viathe automated delivery system to cease the reaction; removing thesolvent; and recovering the at least one cannabinoid or cannabinoidanalog produced by the reaction. Preferably, the reaction mixturecomprises DMSO and the cannabinoid acid synthase is CBDA synthase orTHCA synthase. Even more preferably, the step of reacting thecannabigerol with the cannabinoid acid synthase comprises controllingthe pH of the reaction mixture via a controller. Thus, in a preferredaspect of the invention, the method further comprises controlling the pHof the reaction mixture to produce a predetermined quantity of at leasta first cannabinoid or first cannabinoid analog and controlling the pHof the reaction mixture to produce the predetermined quantity of thefirst cannabinoid or first cannabinoid analog and a predeterminedquantity of a second cannabinoid or second cannabinoid analog. In apreferred embodiment, the first cannabinoid or first cannabinoid analogis THCA or CBDA and the second cannabinoid or second cannabinoid analogis CBCA. In another preferred embodiment, the first cannabinoid or firstcannabinoid analog is THCA or CBDA and the second cannabinoid or secondcannabinoid analog is CBCA.

In another embodiment, the invention provides a method for producing atleast one cannabinoid or cannabinoid analog, that comprises: reactingcannabigerol with cannabinoid acid synthase in a reaction mixturecomprising a solvent and an amphiphilic compound; adding a solvent tocease the reaction; removing the solvent; and recovering the cannabinoidor cannabinoid analog produced by the reaction. In a preferred aspect ofthe invention, the step of reacting the cannabigerol with thecannabinoid acid synthase comprises controlling the pH of the reactionmixture. Preferably, the pH of the reaction mixture is controlled byadjusting the pH of the reaction mixture to achieve a predeterminedratio of a first cannabinoid or first cannabinoid analog to a secondcannabinoid or second cannabinoid analog. Even more preferably, thereaction mixture comprises DMSO and the cannabinoid acid synthase isCBDA synthase or THCA synthase. In one embodiment, the first cannabinoidor first cannabinoid analog is THCA or CBDA and the second cannabinoidor second cannabinoid analog is CBCA.

In an additional embodiment, the invention provides an apparatus thatcomprises an automated supply system configured to deliver a firstautomated supply of cannabigerol, a cannabinoid acid synthase, and areaction mixture comprising a solvent and an amphiphilic compound; abioreactor configured to receive the first supply and permit reaction ofthe cannabigerol and cannabinoid acid synthase in the reaction mixture,and a second automated supply of a solvent so as to cease the reaction;and an extractor configured to remove the solvent and recover at least afirst cannabinoid or cannabinoid analog. The apparatus may furthercomprise a controller configured to adjust at least one property of thereaction mixture so as to produce the first cannabinoid or firstcannabinoid analog and a second cannabinoid or second cannabinoid in apredetermined ratio. The controller may be also configured to determinea first quantity of the first cannabinoid or first cannabinoid analogand a second quantity of a second cannabinoid or second cannabinoidanalog, and adjust at least one property of the reaction mixture so asto produce the first quantity of the first cannabinoid or firstcannabinoid analog and the second quantity of a second cannabinoid orsecond cannabinoid. Preferably, the reaction mixture comprises DMSO andthe cannabinoid acid synthase is CBDA synthase or THCA synthase. In apreferred aspect of the invention, the first cannabinoid or firstcannabinoid analog is THCA or CBDA and the second cannabinoid or secondcannabinoid analog is CBCA.

In yet another embodiment, the invention provides an apparatus forproducing tetrahydrocannabinolic acid (THCA) and cannabichromenic acid(CBCA) or cannabidiolic acid (CBDA) and cannabichromenic acid (CBCA) indifferent ratios comprising: a bioreactor comprising an automated supplysystem configured to deliver (a) a first automated supply ofcannabigerol, a cannabinoid acid synthase, and a reaction mixturecomprising a solvent and an amphiphilic compound, wherein the solvent isone or more of dimethyl sulfoxide (DMSO), dimethyl formamide (DMF) andiso-propoyl alcohol and the concentration of the solvent in the reactionmixture is between 5% and 30% (w/v), and wherein the amphiphiliccompound is a surfactant or a cyclodextrin and the concentration of theamphiphilic compound in the reaction mixture is between 2 and 28 mg/ml;and (b) a second automated supply of solvent to cease the reaction; anextractor configured to remove the solvent and recovertetrahydrocannabinolic acid (THCA) and cannabichromenic acid (CBCA) orcannabidiolic acid (CBDA) and cannabichromenic acid (CBCA) from thereaction mixture; and a controller configured to modify the pH of thereaction mixture to produce tetrahydrocannabinolic acid (THCA) andcannabichromenic acid (CBCA) or cannabidiolic acid (CBDA) andcannabichromenic acid (CBCA) in different ratios, and adjust theconcentration of the amphiphilic compound in the reaction mixture toaffect the conversion rate of cannabigerolic acid (CBGA) intotetrahydrocannabinolic acid (THCA) and cannabichromenic acid (CBCA) orinto cannabidiolic acid (CBDA) and cannabichromenic acid (CBCA) indifferent ratios. In a preferred embodiment, the cannabinoid acidsynthase is tetrahydrocannabinolic acid synthase (THCA synthase) orcannabidiolic acid synthase (CBDA synthase). In one aspect of theinvention, the cannabinoid synthase is immobilized on a solid support.In another aspect of the invention, the cannabinoid synthase is arecombinant cannabinoid synthase, and the apparatus further comprises asystem to produce the recombinant cannabinoid synthase in large scale.Preferably, the pH is in the range from about 3.8 to about 8.0.

In a preferred aspect of the invention, the solvent is one or more ofdimethyl sulfoxide (DMSO), dimethyl formamide (DMF) and iso-propoylalcohol, and the concentration of the solvent in the reaction mixture isbetween 5% and 30% (w/v). In another preferred aspect of the invention,the amphiphilic compound is a surfactant or a cyclodextrin. Thecyclodextrin can be α-cyclodextrin, β-cyclodextrin or γ-cyclodextrin. Inone aspect of the invention, the cyclodextrin is sulfobuthyletherβ-cyclodextrin sodium salt or randomly methylated β-cyclodextrin, andthe concentration of cyclodextrin in the reaction mixture is between 2and 28 mg/ml. Preferably, the concentration of cyclodextrin in thereaction mixture is 8 mg/ml.

In one embodiment, the tetrahydrocannabinolic acid (THCA) andcannabichromenic acid (CBCA) or cannabidiolic acid (CBDA) andcannabichromenic acid (CBCA) are single enantiomers with an enantiomericpurity of at least 95%.

In one aspect of the invention, the cannabinoid synthase is THCAsynthase and the amphiphilic compound is cyclodextrin. Preferably, themass:mass ratio of cyclodextrin to cannabigerolic acid (CBGA) is 28:1 orthe molar ratio of cyclodextrin to cannabigerolic acid (CBGA) is 7.3:1.In a preferred aspect of the invention, the apparatus producestetrahydrocannabinolic acid (THCA) and cannabichromenic acid (CBCA) inthe following ratios:

pH THCA CBCA 4 1 0 5 2.33 1 6 1 5.67 7 0 1

Preferably, 98% of the cannabigerolic acid CBGA is converted intotetrahydrocannabinolic acid (THCA) and cannabichromenic acid (CBCA)within two hours.

In a different aspect of the invention, the cannabinoid acid synthase isCBDA synthase and the amphiphilic compound is cyclodextrin. Preferably,the mass:mass ratio of cyclodextrin to the cannabigerolic acid (CBGA) is11:1 or the molar ratio of cyclodextrin to the cannabigerolic acid(CBGA) is 4:1. In a preferred aspect of the invention, the apparatusproduces cannabidiolic acid (CBDA) and cannabichromenic acid (CBCA) inthe following ratios:

pH CBDA CBCA 4.2 2.5 1 5 1.13 1 5.2 1 1.17 5.4 1 2.45 5.8 1 6.14 6.2 128.13 6.8 0 0

Preferably, 98% of the cannabigerolic acid (CBGA) is converted intocannabidiolic acid (CBDA) and cannabichromenic acid (CBCA) within twohours.

The foregoing general description and the detailed description areexemplary and explanatory and are intended to provide furtherexplanation of the invention as claimed. For detailed understanding ofthe invention, reference is made to the following detailed descriptionof the preferred embodiments, taken in conjunction with the accompanyingdrawing. Other objects, advantages and novel features will be readilyapparent to those skilled in the art from the following detaileddescription of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the effect of 10% DMSO and no cyclodextrin on THCAsynthase activity. 100 μl THCA synthase in crude fermentationsupernatant (10× concentrated) were reacted with 50 μl 2 mg/ml CBGA in350 μl citrate buffer at pH 4.85. Peaks (from left to right): #1 CBGA(86.51%), #2 THCA (10.4%), #3 CBCA (3.09%).

FIG. 2 illustrates the effect of 20% DMSO and no cyclodextrin on THCAsynthase activity. 100 μl THCA synthase in crude fermentationsupernatant (10× concentrated) were reacted with 50 μl 2 mg/ml CBGA in300 μl citrate buffer at pH 4.85 in the presence of 50 μl DMSO. Peaks(from left to right): #1 CBGA (54.18%), #2 THCA (32.39%), #3 CBCA(13.43%).

FIG. 3 illustrates the effect of 10% DMSO and 20% cyclodextrin on THCAsynthase activity. 100 μl THCA synthase in crude fermentationsupernatant (10× concentrated) were reacted with 50 μl 2 mg/ml CBGA in350 μl citrate buffer at pH 4.85 containing 2 mg cyclodextrin. Peaks(from left to right): #1 CBGA (10.33%), #2 THCA (72.37%), #3 CBCA(17.3%).

FIG. 4 illustrates the effect of 10% DMSO and 40% cyclodextrin on THCAsynthase. 150 μl THCA synthase in crude fermentation supernatant (10×concentrated) were reacted with 75 μl 2 mg/ml CBGA in 525 μl citratebuffer at pH 4.85 containing 3 mg cyclodextrin. Peaks (from left toright): #1 CBGA (11.50%), #2 THCA (72.08%), #3 CBCA (16.42%).

FIG. 5 illustrates the effect of 10% DMSO and 60% cyclodextrin on THCAsynthase activity. 200 μl THCA synthase in crude fermentationsupernatant (10× concentrated) were reacted with 100 μl 2 mg/ml CBGA in700 μl citrate buffer at pH 4.85 containing 4 mg cyclodextrin. Peaks(from left to right): #1 CBGA (10.36%), #2 THCA (73.98%), #3 CBCA(15.65%).

FIG. 6 illustrates the effect of 10% DMSO and 20 mg/ml cyclodextrin onCBDA synthase activity. 200 μl CBDA synthase in crude fermentationsupernatant (10× concentrated) were reacted with 100 μl 4 mg/ml CBGA in700 μl citrate buffer at pH 4.85 containing 4 mg cyclodextrin. Peaks(from left to right): #1 CBDA (40.63%), #2 CBGA (28.43%), #3 CBCA(30.95%).

FIG. 7 is a block diagram of a system for producing cannabinoids and/orcannabinoid analogs.

FIG. 8 is a block diagram of a system for producing cannabinoids and/orcannabinoid analogs.

FIG. 9 is a flow diagram illustrating a method for producingcannabinoids.

FIG. 10 is a block diagram of a controller.

FIG. 11 illustrates the effect of 0 mg/ml cyclodextrin on CBDA synthasereaction conversion rate and product ratio. 50 μl of 10× concentratedfermentation supernatant were reacted with 25 μl of 5 mg/ml CBGA in 175μl of citrate buffer pH 4.8. Peaks (Left to right): CBDA (17.99%), CBGA(65.72%), CBCA (16.30%).

FIG. 12 illustrates the effect of 2 mg/ml cyclodextrin on CBDA synthasereaction conversion rate and product ratio. 50 μl of 10× concentratedfermentation supernatant were reacted with 25 μl of 5 mg/ml CBGA in 175μl of citrate buffer pH 4.8 containing 2 mg/ml cyclodextrin. Peaks (Leftto right): CBDA (29.53%), CBGA (47.40%), CBCA (23.08%).

FIG. 13 illustrates the effect of 8 mg/ml cyclodextrin on CBDA synthasereaction conversion rate and product ratio. 50 μl of 10× concentratedfermentation supernatant were reacted with 8.25 μl of 5 mg/ml CBGA in175 μl of citrate buffer pH 4.8 containing 8 mg/ml cyclodextrin. Peaks(Left to right): CBDA (33%), CBGA (41.98%), CBCA (25.02%).

FIG. 14 illustrates the effect of 12 mg/ml cyclodextrin on CBDA synthasereaction conversion rate and product ratio. 50 μl of 10× concentratedfermentation supernatant were reacted with 25 μl of 5 mg/ml CBGA in 175μl of citrate buffer pH 4.8 containing 12 mg/ml cyclodextrin. Peaks(Left to right): CBDA (30.63%), CBGA (45.22%), CBCA (24.15%).

FIG. 15 illustrates the effect of 16 mg/ml cyclodextrin on CBDA synthasereaction conversion rate and product ratio. 50 μl of 10× concentratedfermentation supernatant were reacted with 25 μl of 5 mg/ml CBGA in 175μl of citrate buffer pH 4.8 containing 16 mg/ml cyclodextrin. Peaks(Left to right): CBDA (28.54%), CBGA (49.63%), CBCA (21.84%).

FIG. 16 illustrates the effect of 20 mg/ml cyclodextrin on CBDA synthasereaction conversion rate and product ratio. 50 μl of 10× concentratedfermentation supernatant were reacted with 25 μl of 5 mg/ml CBGA in 175μl of citrate buffer pH 4.8 containing 20 mg/ml cyclodextrin. Peaks(Left to right): CBDA (29.05%), CBGA (50.04%), CBCA (20.91%).

FIG. 17 illustrates the effect of 28 mg/ml cyclodextrin on CBDA synthasereaction conversion rate and product ratio. 50 μl of 10× concentratedfermentation supernatant were reacted with 25 μl of 5 mg/ml CBGA in 175μl of citrate buffer pH 4.8 containing 28 mg/ml cyclodextrin. Peaks(Left to right): CBDA (22.09%), CBGA (59.60%), CBCA (18.32%).

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a system and methods for large scalesimultaneous enzymatic production of different cannabinoids orcannabinoid analogs, as well as methods for cloning, expressing andpurifying enzymes that catalyze large scale simultaneous synthesis ofTHCA, CBDA, CBCA or analogs thereof under various pH, temperature andaqueous/lipophilic conditions.

Definitions

As used herein, unless otherwise stated, the singular forms “a,” “an,”and “the” include plural reference. Thus, for example, a reference to “acell” includes a plurality of cells, and a reference to “a molecule” isa reference to one or more molecules.

As used herein, “about” will be understood by persons of ordinary skillin the art and will vary to some extent depending upon the context inwhich it is used. If there are uses of the term which are not clear topersons of ordinary skill in the art, given the context in which it isused, “about” will mean up to plus or minus 10% of the particular term.

The term “alkyl” refers to a straight or branched chain, saturatedhydrocarbon having the indicated number of carbon atoms. For example,(C₁-C₁₀) alkyl is meant to include but is not limited to methyl, ethyl,propyl, isopropyl, butyl, sec-butyl, tert-butyl, pentyl, isopentyl,neopentyl, hexyl, isohexyl, and neohexyl, etc. An alkyl group can beunsubstituted or optionally substituted with one or more substituents asdescribed herein below.

The term “alkenyl” refers to a straight or branched chain unsaturatedhydrocarbon having the indicated number of carbon atoms and at least onedouble bond. Examples of a (C₂-C₁₀) alkenyl group include, but are notlimited to, ethylene, propylene, 1-butylene, 2-butylene, isobutylene,sec-butylene, 1-pentene, 2-pentene, isopentene, 1-hexene, 2-hexene,3-hexene, isohexene, 1-heptene, 2-heptene, 3-heptene, isoheptene,1-octene, 2-octene, 3-octene, 4-octene, and isooctene. An alkenyl groupcan be unsubstituted or optionally substituted with one or moresubstituents as described herein below.

The term “alkynyl” refers to a straight or branched chain unsaturatedhydrocarbon having the indicated number of carbon atoms and at least onetriple bond. Examples of a (C₂-C₁₀) alkynyl group include, but are notlimited to, acetylene, propyne, 1-butyne, 2-butyne, 1-pentyne,2-pentyne, 1-hexyne, 2-hexyne, 3-hexyne, 1-heptyne, 2-heptyne,3-heptyne, 1-octyne, 2-octyne, 3-octyne and 4-octyne. An alkynyl groupcan be unsubstituted or optionally substituted with one or moresubstituents as described herein below.

The term “alkoxy” refers to an —O-alkyl group having the indicatednumber of carbon atoms. For example, a (C₁-C₆) alkoxy group includes—O-methyl, —O-ethyl, —O-propyl, —O-isopropyl, —O-butyl, —O-sec-butyl,—O-tert-butyl, —O-pentyl, —O-isopentyl, —O-neopentyl, —O-hexyl,—O-isohexyl, and —O-neohexyl.

The term “aryl” refers to a 3- to 14-member monocyclic, bicyclic,tricyclic, or polycyclic aromatic hydrocarbon ring system. Examples ofan aryl group include naphthyl, pyrenyl, and anthracyl. An aryl groupcan be unsubstituted or optionally substituted with one or moresubstituents as described herein below.

The terms “alkylene,” “alkenylene,” and “arylene,” alone or as part ofanother substituent, means a divalent radical derived from an alkyl,cycloalkyl, alkenyl, aryl, or heteroaryl group, respectively, asexemplified by —CH₂CH₂CH₂CH₂—. For alkylene, alkenyl, or aryl linkinggroups, no orientation of the linking group is implied.

The term “halogen” and “halo” refers to —F, —Cl, —Br or —I.

The term “heteroatom” is meant to include oxygen (O), nitrogen (N), andsulfur (S).

A “hydroxyl” or “hydroxy” refers to an —OH group.

The term “hydroxyalkyl,” refers to an alkyl group having the indicatednumber of carbon atoms wherein one or more of the alkyl group's hydrogenatoms is replaced with an —OH group. Examples of hydroxyalkyl groupsinclude, but are not limited to, —CH₂OH, —CH₂CH₂OH, —CH₂CH₂CH₂OH,—CH₂CH₂CH₂CH₂OH, —CH₂CH₂CH₂CH₂CH₂OH, —CH₂CH₂CH₂CH₂CH₂CH₂OH, and branchedversions thereof.

The term “cycloalkyl” refer to monocyclic, bicyclic, tricyclic, orpolycyclic, 3- to 14-membered ring systems, which are either saturated,unsaturated or aromatic. The heterocycle may be attached via anyheteroatom or carbon atom. Cycloalkyl include aryls and hetroaryls asdefined above. Representative examples of cycloalky include, but are notlimited to, cycloethyl, cyclopropyl, cycloisopropyl, cyclobutyl,cyclopentyl, cyclohexyl, cyclopropene, cyclobutene, cyclopentene,cyclohexene, phenyl, naphthyl, anthracyl, benzofuranyl, andbenzothiophenyl. A cycloalkyl group can be unsubstituted or optionallysubstituted with one or more substituents as described herein below.

The term ‘nitrile or cyano” can be used interchangeably and refer to a—CN group which is bound to a carbon atom of a heteroaryl ring, arylring and a heterocycloalkyl ring.

The term “amine or amino” refers to an —NR_(c)R_(d) group wherein R_(c)and R_(d) each independently refer to a hydrogen, (C₁-C₅)alkyl, aryl,heteroaryl, heterocycloalkyl, (C₁-C₅)haloalkyl, and (C₁-C₆)hydroxyalkylgroup.

The term “alkylaryl” refers to C₁-C₈ alkyl group in which at least onehydrogen atom of the C₁-C₈ alkyl chain is replaced by an aryl atom,which may be optionally substituted with one or more substituents asdescribed herein below. Examples of alkylaryl groups include, but arenot limited to, methylphenyl, ethylnaphthyl, propylphenyl, andbutylphenyl groups.

“Arylalkylene” refers to a divalent alkylene wherein one or morehydrogen atoms in the C₁-C₁₀ alkylene group is replaced by a(C₃-C₁₄)aryl group. Examples of (C₃-C₁₄)aryl-(C₁-C₁₀)alkylene groupsinclude without limitation 1-phenylbutylene, phenyl-2-butylene,1-phenyl-2-methylpropylene, phenylmethylene, phenylpropylene, andnaphthylethylene.

“Arylalkenylene” refers to a divalent alkenylene wherein one or morehydrogen atoms in the C₂-C₁₀ alkenylene group is replaced by a(C₃-C₁₄)aryl group.

The term “arylalkynylene” refers to a divalent alkynylene wherein one ormore hydrogen atoms in the C₂-C₁₀ alkynylene group is replaced by a(C₃-C₁₄)aryl group.

The terms “carboxyl” and “carboxylate” include such moieties as may berepresented by the general formulas:

E in the formula is a bond or O and R^(f) individually is H, alkyl,alkenyl, aryl, or a pharmaceutically acceptable salt. Where E is O, andR^(f) is as defined above, the moiety is referred to herein as acarboxyl group, and particularly when R^(f) is a hydrogen, the formularepresents a “carboxylic acid”. In general, where the expressly shownoxygen is replaced by sulfur, the formula represents a “thiocarbonyl”group.

Unless otherwise indicated, “stereoisomer” means one stereoisomer of acompound that is substantially free of other stereoisomers of thatcompound. Thus, a stereomerically pure compound having one chiral centerwill be substantially free of the opposite enantiomer of the compound. Astereomerically pure compound having two chiral centers will besubstantially free of other diastereomers of the compound. A typicalstereomerically pure compound comprises greater than about 80% by weightof one stereoisomer of the compound and less than about 20% by weight ofother stereoisomers of the compound, for example greater than about 90%by weight of one stereoisomer of the compound and less than about 10% byweight of the other stereoisomers of the compound, or greater than about95% by weight of one stereoisomer of the compound and less than about 5%by weight of the other stereoisomers of the compound, or greater thanabout 97% by weight of one stereoisomer of the compound and less thanabout 3% by weight of the other stereoisomers of the compound.

The present invention provides methods for the enzymatic synthesis ofcannabinoids or cannabinoid analogs in a cell-free environment. Alsodescribed is an apparatus for the ex vivo manufacture of cannabinoidsand analogs of cannabinoids. The term “analog” refers to a compound thatis structurally related to naturally occurring cannabinoids, but whosechemical and biological properties may differ from naturally occurringcannabinoids. In the present context, analog or analogs refer compoundsthat may not exhibit one or more unwanted side effects of a naturallyoccurring cannabinoid. Analog also refers to a compound that is derivedfrom a naturally occurring cannabinoid by chemical, biological or asemi-synthetic transformation of the naturally occurring cannabinoid.

Cannabinoid compounds include, but are not limited to, cannabinol,cannabidiol, Δ9-tetrahydrocannabinol, Δ8-tetrahydrocannabinol,11-hydroxy-tetrahydrocannabinol, 11-hydroxy-Δ9-tetrahydrocannabinol,levonantradol, Δ11-tetrahydrocannabinol, tetrahydrocannabivarin,dronabinol, amandamide and nabilone, as well as natural or syntheticmolecules that have a basic cannabinoid structure and are modifiedsynthetically to provide a cannabinoid analog.

The present invention also provides methods for the large scale cloningand expression of the enzymes that play a role in the biosynthesis ofcannabinoids and for the use of an eukaryotic expression system for theproduction of biosynthetic enzymes that can be used for the manufactureof cannabinoids and cannabinoid analogs. Yeast as well as eukaryotic andprokaryotic cells are suitable for the cloning and expression of thecannabinoid acid synthase enzymes and include without limitation E.coli, yeast and baculovirus hosts. Thus, the present invention disclosesa method for the large-scale production of several cannabinoid acidsynthase enzymes including, but not limited to, tetrahydrocannabinolicacid (THCA) synthase and cannabidiolic acid (CBDA) synthase, using thepink Pichia yeast expression system. Accordingly, large scale productionof these enzymes can be carried out by transforming yeast with a DNAconstruct that comprises a gene for a cannabinoid synthase, generatingone or more copies of the cannabinoid acid synthase gene andoverexpressing a protein encoded by the cannabinoid acid synthase gene.

The nucleic acid sequence of the THCA synthase gene is represented bySEQ ID NO: 1 and encodes a polypeptide sequence set forth in SEQ ID NO:2. The codon optimized nucleic acid sequence of the THCA synthase genefor Pichia pastoris expression is represented by SEQ ID NO: 3 andencodes a polypeptide sequence set forth in SEQ ID NO: 4, which is theTHCA synthase amino acid sequence comprising the alpha secretionsequence of Pichia pastoris. “THCA synthase expression” refers to thebiosynthesis of a gene product encoded by SEQ ID NO: 1 or by SEQ ID NO:3, or a variant, fragment or portion of SEQ ID NO: 1 or SEQ ID NO: 3.“THCA synthase expression” also refers to the biosynthesis of apolypeptide comprising SEQ ID NO: 2 or SEQ ID NO: 4, or a variant,fragment or portion of a polypeptide comprising SEQ ID NO: 2 or SEQ IDNO: 4. “THCA synthase overexpression” denotes an increase in THCAsynthase expression. THCA overexpression affects an increase in THCA orCBCA content for a plant or cell in which the overexpression occurs.THCA overexpression refers to upregulated biosynthesis of a gene productencoded by SEQ ID NO: 1 or by SEQ ID NO: 3, or any variant, fragment orportion of SEQ ID NO: 1 or SEQ ID NO: 3.

The nucleic acid sequence of the CBDA synthase gene (codon optimized forPichia pastoris expression) is represented by SEQ ID NO: 5 and encodes apolypeptide sequence set forth in SEQ ID NO: 6. The codon optimizednucleic acid sequence of the CBDA synthase gene for Pichia pastorisexpression is represented by SEQ ID NO: 7 and encodes a polypeptidesequence set forth in SEQ ID NO: 8, which is the CBDA synthase aminoacid sequence comprising the alpha secretion sequence of Pichiapastoris. “CBDA synthase expression” refers to the biosynthesis of agene product encoded by SEQ ID NO: 5 or by SEQ ID NO: 7, or a variant,fragment or portion of SEQ ID NO: 5 or SEQ ID NO: 7. “CBDA synthaseexpression” also refers to the biosynthesis of a polypeptide comprisingSEQ ID NO: 6 or SEQ ID NO: 8, or a variant, fragment or portion of apolypeptide comprising SEQ ID NO: 6 or SEQ ID NO: 8. “CBDA synthaseoverexpression” denotes an increase in CBDA synthase expression. CBDAoverexpression affects an increase in CBDA or CBCA content for a plantor cell in which the overexpression occurs. CBDA overexpression refersto upregulated biosynthesis of a gene product encoded by SEQ ID NO: 5 orby SEQ ID NO: 7, or any variant, fragment or portion of SEQ ID NO: 5 orSEQ ID NO: 7.

The present invention encompasses any nucleic acid, gene,polynucleotide, DNA, RNA, mRNA, or cDNA molecule that is isolated fromthe genome of a plant species, or produced synthetically, that increasesbiosynthesis of cannabinoids or cannabinoid analogs. Additionally,expression of such cannabinoid acid synthase sequence producescannabinoids or cannabinoid analogs in a non-cannabinoid producing cell,including yeast, prokariotic cells and eukariotic cells, such as anon-cannabinoid producing plant cell, a bacteria cell, an insect cell,or an yeast cell. The DNA or RNA may be double-stranded orsingle-stranded. Single-stranded DNA may be the coding strand, alsoknown as the sense strand, or it may be the non-coding strand, alsocalled the anti-sense strand.

It is understood that THCA synthase and CBDA synthase include thesequences set forth in SEQ ID NOs: 1, 3, 5 and 7, respectively, as wellas nucleic acid molecules comprising variants, fragments or portions ofSEQ ID NOs: 1, 3, 5 and 7, with one or more bases deleted, substituted,inserted, or added, wherein a variant of anyone of SEQ ID Nos: 1, 3, 5and 7 codes for a polypeptide with cannabinoid or cannabinoid analogbiosynthesis activity. Accordingly, sequences having “base sequenceswith one or more bases deleted, substituted, inserted, or added” retainphysiological activity even when the encoded amino acid sequence has oneor more amino acids substituted, deleted, inserted, or added.Physiological activity of the encoded amino acid sequences may be testedusing conventional enzymatic assays known in the art. Additionally,multiple forms of THCA synthase and CBDA synthase may exist, which maybe due to post-translational modification of a gene product, or tomultiple forms of the respective THCA synthase and CBDA synthase.Nucleotide sequences that have such modifications and that code forcannabinoid or cannabinoid analog biosynthesis enzymes are includedwithin the scope of the present invention.

For example, the poly A tail or 5′- or 3′-end, nontranslation regionsmay be deleted, and bases may be deleted to the extent that amino acidsare deleted. Bases may also be substituted, as long as no frame shiftresults. Bases also may be “added” to the extent that amino acids areadded. It is essential, however, that any such modification does notresult in the loss of cannabinoid acid or cannabinoid acid analogbiosynthesis enzyme activity. A modified DNA in this context can beobtained by modifying the DNA base sequences of the invention so thatamino acids at specific sites are substituted, deleted, inserted, oradded by site-specific mutagenesis, for example, and that still retaincannabinoid acid or cannabinoid acid analog biosynthesis enzymeactivity. Cannabinoid acid or cannabinoid acid analog biosynthesisenzyme activity of the encoded amino acid sequences may be assayed asdescribed above.

A cannabinoid or cannabinoid analog biosynthesis sequence can besynthesized ab initio from the appropriate bases, for example, by usingan appropriate protein sequence disclosed herein as a guide to create aDNA molecule that, though different from the native DNA sequence,results in the production of a protein with the same or similar aminoacid sequence. This type of synthetic DNA molecule is useful whenintroducing a DNA sequence into a non-plant cell, coding for aheterologous protein, that reflects different (non-plant) codon usagefrequencies and, if used unmodified, can result in inefficienttranslation by the host cell.

By “isolated” nucleic acid molecule(s) is intended a nucleic acidmolecule, DNA or RNA, which has been removed from its nativeenvironment. For example, recombinant DNA molecules contained in a DNAconstruct are considered isolated for the purposes of the presentinvention. Further examples of isolated DNA molecules includerecombinant DNA molecules maintained in heterologous host cells or DNAmolecules that are purified, partially or substantially, in solution.Isolated RNA molecules include in vitro RNA transcripts of the DNAmolecules of the present invention. Isolated nucleic acid molecules,according to the present invention, further include such moleculesproduced synthetically.

“Exogenous nucleic acid” refers to a nucleic acid, DNA or RNA, which hasbeen artificially introduced into a cell. Such exogenous nucleic acidmay be a copy of a sequence which is naturally found in the cell intowhich it was introduced, or fragments thereof.

In contrast, “endogenous nucleic acid” refers to a nucleic acid, gene,polynucleotide, DNA, RNA, mRNA, or cDNA molecule that is present in thegenome of a plant or organism that is to be genetically engineered. Anendogenous sequence is “native” to, i.e., indigenous to, the plant ororganism that is to be genetically engineered.

“Heterologous nucleic acid” refers to a nucleic acid, DNA or RNA, whichhas been introduced into a cell which is not a copy of a sequencenaturally found in the cell into which it is introduced. Suchheterologous nucleic acid may comprise segments that are a copy of asequence which is naturally found in the cell into which it has beenintroduced, or fragments thereof.

A “chimeric nucleic acid” comprises a coding sequence or fragmentthereof linked to a transcription initiation region that is differentfrom the transcription initiation region with which it is associated incells in which the coding sequence occurs naturally.

The present application is directed to such nucleic acid molecules whichare at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%or 100% identical to a nucleic acid sequence described in any of SEQ IDNO: 1, 3, 5 and 7. Preferred are nucleic acid molecules which are atleast 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleic acidsequence shown in any of SEQ ID NO: 1, 3, 5 and 7. Differences betweentwo nucleic acid sequences may occur at the 5′ or 3′ terminal positionsof the reference nucleotide sequence or anywhere between those terminalpositions, interspersed either individually among nucleotides in thereference sequence or in one or more contiguous groups within thereference sequence.

As a practical matter, whether any particular nucleic acid molecule isat least 95%, 96%, 97%, 98% or 99% identical to a reference nucleotidesequence refers to a comparison made between two molecules usingstandard algorithms well known in the art and can be determinedconventionally using publicly available computer programs such as theBLAST algorithm.

The present invention further provides nucleic acid molecules comprisingthe nucleotide sequence of SEQ ID NOs: 1, 3, 5 and 7, respectively,which encode an active cannabinoid or cannabinoid analog biosynthesisenzyme, wherein the enzyme has an amino acid sequence that correspondsto SEQ ID NOs: 2, 4, 6, and 8, respectively, or a variant, fragment orportion of anyone of SEQ ID Nos: 2, 4, 6 and 8, and wherein the proteinof the invention encompasses amino acid substitutions, additions anddeletions that do not alter the function of the cannabinoid orcannabinoid analog biosynthesis enzyme.

A “variant” is a nucleotide or amino acid sequence that deviates fromthe standard, or given, nucleotide or amino acid sequence of aparticular gene or protein. The terms “isoform,” “isotype,” and “analog”also refer to “variant” forms of a nucleotide or an amino acid sequence.An amino acid sequence that is altered by the addition, removal, orsubstitution of one or more amino acids, or a change in nucleotidesequence, may be considered a “variant” sequence. The variant may have“conservative” changes, wherein a substituted amino acid has similarstructural or chemical properties, e.g., replacement of leucine withisoleucine. A variant may have “nonconservative” changes, e.g.,replacement of a glycine with a tryptophan. Analogous minor variationsmay also include amino acid deletions or insertions, or both. Guidancein determining which amino acid residues may be substituted, inserted,or deleted may be found using computer programs well known in the art.

The invention contemplates genetically engineering “non-cannabinoid orcannabinoid analog producing cells” with a nucleic acid sequenceencoding an enzyme involved in the production of cannabinoids orcannabinoid analogs. Non-cannabinoid or cannabinoid analog producingcells refer to a cell from any organism that does not produce acannabinoid or cannabinoid analog. Illustrative cells include but arenot limited to plant cells, as well as insect, mammalian, yeast, fungal,algal, or bacterial cells.

“Fungal cell” refers to any fungal cell that can be transformed with agene encoding a cannabinoid or cannabinoid analog biosynthesis enzymeand is capable of expressing in recoverable amounts the enzyme or itsproducts. Illustrative fungal cells include yeast cells such asSaccharomyces cerivisae and Pichia pastoris. Cells of filamentous fungisuch as Aspergillus and Trichoderma may also be used.

Cannabinoid acid synthase gene sequences may be obtained from a publiclyavailable database. In a preferred aspect of the invention, one or morecopies of a cannabinoid acid synthase gene are generated in vivo and themethod comprises integrating one or more copies of the cannabinoid acidsynthase gene into the genome of a eukaryotic host, such as Pichiapastoris, to scale up protein expression. Preferably, the cannabinoidacid synthase gene is codon optimized with an alpha secretion sequenceto target protein secretion or tagged with six tandem histidine (SEQ IDNO: 9) residues at the 3′ end to facilitate purification. This processcomprises linearizing the cannabinoid acid synthase gene by digestionwith one or more restriction enzymes; extracting the cannabinoid acidsynthase gene by gel extraction; ligating the cannabinoid acid synthasegene into a Pichia pastoris plasmid; and electroporating the plasmidinto bacterial cells to generate one or more cannabinoid acid synthasegene copy colonies.

Thus, in one embodiment, one or more copies of alpha-CBDA synthase andalpha-THCA synthase sequences, for example, are generated bymodification as described above, insertion into pPink-HC vector(Invitrogen®) and transformation into E. coli cells. The transformedcells may be stored as agar stabs for future use. Prior totransformation of yeast cells, the vector containing the cannabinoidacid synthase gene of interest (GOI) is isolated from the agar stabscontaining the transformed E. coli cells, linearized using PmeI or Spelrestriction enzymes and the linearized plasmids thus obtained areelectroporated into Pichia pastoris pepB deficient mutant cells usingPichiaPink™ Yeast Expression Systems (Invitrogen®). Linearization withthe restriction enzyme PmeI directs the insert into the AOX1 promoterregion of the Pichia genome, whereas linearization with the restrictionenzyme Spel directs the insert into the TRP gene.

The transformed yeast cells may be grown on adenine-deficient selectiveplates and the colonies thus formed may be screened to identify positivetransformants. Screening methods include, but are not limited to, colorscreening methodology. Typically, cells having 6-10 copies of the geneof interest are desired for obtaining large amounts of recombinantprotein, for example, about 1.0 g to about 2.0 g of protein per liter ofculture.

In one embodiment, individual white colonies of yeast cells carrying theTHCA synthase gene or the CBDA synthase gene, for example, areseparately cultured in flasks using BMGY medium, followed by inductionby growth in BMMY medium, to induce the expression of THCA synthase orCBDA synthase as further described below. Briefly, the medium containingthe enzyme in each culture is separated from the cells, reacted with aknown amount of substrate and the product is analyzed. Cultures oftransformants showing greater than 20% conversion are used for thecommercial synthesis of cannabinoids or cannabinoid analogs pursuant tomethods of the invention.

The cannabinoid acid synthase enzymes, THCA synthase and CBDA synthase,obtained using the PichiaPink™ Yeast Expression system described above,can be used for the manufacture of cannabinoids or cannabinoid analogs.The cannabinoid or cannabinoid analogs thus obtained are isolated,purified and used as therapeutics. In a further embodiment, thecannabinoids or cannabinoid analogs thus obtained undergo adecarboxylation step.

Cannabinoid synthases according to the invention include, but are notlimited to, cannabidiolic acid (CBDA) synthase andtetrahydrocannabinolic acid (THCA) synthase.

In one embodiment, the invention provides a method for producing acannabinoid or a cannabinoid analog by selecting a Formula I compoundand a cannabinoid acid synthase as a catalyst for transforming theFormula I compound to a cannabinoid or a cannabinoid analog.

In Formula I, R can be selected from hydroxyl (—OH), halogen, thiol(—SH), or a —NR_(a)R_(b) group. Substituent groups R₁ and R₂ are eachindependently selected from the group consisting of —H, —C(O)R_(a),—OR_(a), an optionally substituted C₁-C₁₀ linear or branched alkylene,an optionally substituted C₂-C₁₀ linear or branched alkenylene, anoptionally substituted C₂-C₁₀ linear or branched alkynylene, anoptionally substituted C₃-C₁₀ aryl, an optionally substituted C₃-C₁₀cycloalkyl, (C₃-C₁₀)aryl-(C₁-C₁₀)alkylene,(C₃-C₁₀)aryl-(C₂-C₁₀)alkenylene, and (C₃-C₁₀)aryl-(C₁-C₁₀)alkynylene.Alternatively, R₁ and R₂ together with the carbon atoms to which theyare bonded form a C₅-C₁₀ cyclic ring. For compounds according to FormulaI, R₃ is selected from the group consisting of H, —C(O)R_(a) and C₁-C₁₀linear or branched alkyl and R_(a) and R_(b) are each independently —H,—OH, (C₁-C₁₀) linear or branched alkyl, —SH, —NH₂, or a C₃-C₁₀cycloalkyl.

R₂ can be a linear alkylene or a branched alkylene. Exemplary of linearalkylenes include without limitation CH₃, C₂H₅, C₃H₇, C₄H₉, C₅H₁₁,C₆H₁₃, C₇H₁₅ and C₈H₁₇. Illustrative of branched alkylenes are groupsselected from, iso-propyl, sec-butyl, iso-butyl, neopentyl, 2-methylhexyl, or 2,3-dimethyl hexyl groups. In some embodiments, R₂ can be anoptionally substituted linear or branched alkylene in which one or morehydrogen atoms is replaced without limitation with a group selected fromchlorine, fluorine, bromine, nitro, amino, hydroxyl, phenyl, or benzylgroup.

In one embodiment, R₁ and R₂ together with the ring carbon atoms towhich they are bonded form a C₅-C₁₀ cyclic ring. For such Formula Icompounds one or more carbon atoms of the ring can be substituted with aheteroatom selected from oxygen, sulfur or nitrogen.

In another embodiment, R₂ is a C₂-C₁₀ alkenylene and is selected fromthe group consisting of

with R₄ being a linear or branched alkylene as described above. When R₂is a C₂-C₁₀ linear or branched alkynylene, R₂ can be

Alternatively, R₂ in Formula I is

substituent X is a group selected from—OH, —SH, or NR_(a)R_(b) and groups R_(a) and R_(b). are as definedabove.

In one embodiment, the cannabinoids and/or cannabinoid analogssynthesized according to the invention have a carboxylic acid (—COOH)group as the R₁ substituent and may undergo an optional decarboxylationstep prior to their use as pharmaceutical or nutraceutical agents.Examples of cannabinoids or cannabinoid analogs having a carboxylic acidgroup include, but are not limited to, compounds obtained by reacting acompound of Formula I in which R is —OH, R₁ is —COOH, R₂ is C₅H₁₁ and R₃is —H with a cannabinoid acid synthase obtained as described above.

The synthesis, isolation and purification of cannabinoids or cannabinoidanalogs can be improved by immobilization of a cannabinoid acid synthaseto a solid support, or by encapsulation of the synthase within aliposome. In one aspect, the enzyme is immobilized to a solid support.Without being bound to any theory, the inventors of the presentapplication have unexpectedly discovered that immobilization facilitatesuse and recovery of the enzyme catalyst, purification of the desiredproduct, and preservation of the enantiomeric excess (ee) of the finalproduct, and provides an overall improvement in the yield of theproduct. Furthermore, immobilization permits recycling and reuse of theimmobilized enzyme which significantly reduces the costs associated withthe manufacture of pharmaceutical grade cannabinoids or cannabinoidanalogs. Typically, the enantiomeric purity of the cannabinoids and/orcannabinoid analogs produced according to the invention is from about90% ee to about 100% ee, for instance, a cannabinoid or a cannabinoidanalog produced using the inventive methodology can have an enantiomericpurity of about 91% ee, about 92% ee, about 93% ee, about 94% ee, about95% ee, about 96% ee, about 97% ee, about 98% ee and about 99% ee.

Typically, the enzyme to be immobilized can be absorbed onto a solidsupport, adsorbed onto a support, covalently linked to a support or canbe immobilized onto a solid support through ionic interactions. In oneembodiment, the cannabinoid acid synthase is covalently linked to asolid support. Suitable strategies for linking an enzyme to a solidsupport are well known in the biochemical art and include covalentlinkages between an appropriately functionalized support and a sidechain of an amino acid group or through covalent linkages usingappropriately functionalized linkers or spacers to separate the supportfrom the enzyme. The term “linker” refers to any group that separatesthe support from the enzyme. Accordingly, a linker is a group that iscovalently tethered at one end to a group on the surface of the supportand is attached to the enzyme at the other end. Illustrative linkersinclude (C₁-C₁₀)alkylene linker polymers of ethylene glycol such as a—(OCH₂—CH₂)_(n)—O— group, where n is an integer from 0 to 10,—(C₁-C₁₀)alkylene-NH—, —(C₁-C₁₀)alkylenesiloxy, or a—(C₁-C₁₀)alkylene-C(O)—.

Supports suitable for immobilizing enzymes include, but are not limitedto, Amberlite resins, Duolite resins, acrylic resins such as Eupergit®C, DEAE-Sephadex and gels made using polyvinyl alcohol.

Cannabinoids exert different physiological properties and are known tolessen pain, stimulate appetite and have been tested as candidatetherapeutics for treating a variety of disease conditions such asallergies, inflammation, infection, epilepsy, depression, migraine,bipolar disorders, anxiety disorder, and glaucoma. The physiologicaleffects exerted by cannabinoids is affected by their ability tostimulate or deactivate the cannabinoid receptors, for instance the CB1,CB2 and CB3 receptors. Accordingly, the present invention provides themeans to modulate cannabinoid receptor activity and their pharmaceuticalproperties by modifying the cannabinoid and/or cannabinoid analogbinding interactions and the orientation of a ligand within thecannabinoid receptors active site by determining the nature andorientation of substituent groups attached to the cannabinoids and/orcannabinoid analogs produced according to the invention.

Thus, in one embodiment the invention provides a method for themanufacture of cannabinoids and cannabinoid analogs that havestructurally distinct and diverse substituent groups attached to acentral core and thus exhibit different pharmaceutically beneficialproperties. Structural diversity is accomplished by contacting anappropriately substituted Formula III compound with a Formula IVcompound in the presence of an enzyme, such as GPP olivetolategeranyltransferase (a polyketide synthase), to produce a compound ofFormula II. Scheme 1 below structurally illustrates the protocol forsynthesizing a Formula II compound pursuant to this embodiment.

Different compounds of Formula II that serve as substrates for themanufacture of cannabinoids and/or cannabinoid analogs according to theinvention may be obtained by varying the nature and type of substituentgroups at R, R₁, R₂, R₃ and R₅, in the compounds of Formulas III and IV.According to this embodiment, therefore, different cannabinoids and/orcannabinoid analogs may be obtained by reacting a compound of Formula IIwith a cannabinoid acid synthase, for example, THCA synthase or CBDAsynthase obtained as described above, followed by isolation anddecarboxylation of the obtained product to give a cannabinoid or acannabinoid analog.

In Formula III, R can be selected from hydroxyl (—OH), halogen, thiol(—SH), or a —NR_(a)R_(b) group. Substituents R₁ and R₂ are eachindependently selected from the group consisting of —H, —C(O)R_(a),—OR_(a), an optionally substituted linear or branched (C₁-C₁₀)alkylene,an optionally substituted linear or branched (C₂-C₁₀)alkenylene, anoptionally substituted linear or branched (C₂-C₁₀)alkynylene, anoptionally substituted C₃-C₁₀ aryl, an optionally substituted C₃-C₁₀cycloalkyl, (C₃-C₁₀)aryl-(C₁-C₁₀)alkylene,(C₃-C₁₀)aryl-(C₂-C₁₀)alkenylene, and (C₃-C₁₀)aryl-(C₁-C₁₀)alkynylene.

In certain embodiments R₁ and R₂ together with the carbon atoms to whichthey are bonded form a C₅-C₁₀ cyclic ring and R₃ is selected from thegroup consisting of H, —C(O)R_(a) and C₁-C₁₀ linear or branched alkyl.

R₅ in Formula IV can be a linear or branched (C₁-C₁₀)alkylene, a linearor branched (C₂-C₁₀)alkenylene, a linear or branched (C₂-C₁₀)alkynylene,—C(O)— (C₁-C₁₀)alkylene, —C(O)—(C₂-C₁₀)alkenylene and —C(O)—(C₂-C₁₀)alkynylene. For Formulae II, III and IV compounds any alkylene,alkenylene, alkynylene, aryl, arylalkylene, or cycloalkyl group can befurther substituted with one or more groups selected from the groupconsisting of —OH, halogen, —NR_(b)R_(c), —C(O)R_(a), —C(O)NR_(b)R_(c),(C₁-C₁₀)alkyl, —CN, (C₁-C₄)alkoxy, (C₁-C₄)haloalkyl, and(C₁-C₄)hydroxyalkyl with R_(a), R_(b) and R_(c) each independently beingselected from —H, —OH, or (C₁-C₁₀) linear or branched alkyl, —SH, —NH₂,or a C₃-C₁₀ cycloalkyl.

According to one embodiment, R₅ in Formula IV can be a(C₂-C₁₀)alkenylene selected from

with R₄ being a linear alkylene selected from the group consisting ofCH₃, C₂H₅, C₃H₇, C₄H₉, C₅H₁₁, C₆H₁₃, C₇H₁₅ and C₈H₁₇. For certainFormula IV compounds R₅ is

and group R₆ is selected from (C₁-C₁₀)alkylene, (C₂-C₁₀)alkenylene, —OH,—SH, NO₂, F, Cl, Br, —NH₂, or a —NHR_(a) where R_(a) is as definedabove.

A recombinant cannabinoid acid synthase obtained by overexpressing aprotein encoded by a recombinant cannabinoid acid synthase gene asdescribed above is reacted with a substrate according to Formula I orwith a substrate according to Formula II as described above in areaction mixture comprising a solvent and an amphiphilic compound toproduce one or more cannabinoids or cannabinoid analogs. Thecannabinoids or cannabinoid analogs thus formed are isolated from thereaction mixture and optionally decarboxylated. Preferably, therecombinant cannabinoid acid synthase is a recombinant CBDA synthase ora recombinant THCA synthase obtained by the method described above. In apreferred aspect of the invention, the solvent in the reaction mixtureis a non-aqueous solvent, such as dimethyl sulfoxide (DMSO), dimethylformamide (DMF), or iso-propoyl alcohol. The concentration of thesolvent in the reaction mixture may vary between 10% and 30% (v/v). Theinventors of the present application have unexpectedly discovered thatthe concentration of the non-aqueous solvent in the reaction mixtureaffects the rate of the reaction as well as the ratio between thedifferent cannabinoid products. Thus, the table below shows that in areaction driven by the THCA synthase, the presence of DMSO in aconcentration of 20% (v/v) in the reaction mixture increases the rate ofthe reaction by 2.5-fold and causes the reaction to produce THCA andCBCA in a ratio of 5:1, whereas the presence of DMSO in a concentrationof 10% (v/v) in the reaction mixture produces THCA and CBCA in a ratioof 10:1. Accordingly, in a preferred aspect of the invention, thenon-aqueous solvent in the reaction mixture is DMSO and theconcentration of DMSO in the reaction mixture is most preferably 20%(v/v).

TABLE 1 Effect of DMSO Concentration on Reaction Rate and Products DMSORate of Reaction THCA:CBCA  0%  1X 10% 1.2X 10:1  20% 2.5X 5:1 25% — 1:130% 0.3X

In an additional preferred embodiment of the invention, the reactionmixture also comprises an amphiphilic compound. Preferably, theamphiphilic compound is a surfactant or a cyclodextrin. Surfactants mayinclude, but are not limited to, cationic surfactants, ionic surfactantsand anionic surfactants. Most preferably, the reaction mixture containsa cyclodextrin.

Cyclodextrins are natural cyclic oligosaccharides consisting of six ormore 1-4 linked α-anhydro-glucose moieties, which may be produced fromstarch through an enzymatic reaction. Cyclodextrins are classifiedaccording to the number of glucose units as α-cyclodextrin (six units),β-cyclodextrin (seven units) and γ-cyclodextrin (eight units). Thestructure of the cyclodextrin is shown below:

The secondary hydroxyl groups on the exterior side of the cyclodextrinmolecule are hydrophilic, whereas the primary hydroxyl groups form thehydrophobic central cavity. Without being bound to any theory, it isbelieved that the hydrophobic central cavity in cyclodextrinincorporates the substrate in the reaction mixture as a guest moleculeand the complex thus formed protects and stabilizes the substrate,although no covalent or ionic bonds are formed.

The inventors of the present application have unexpectedly discoveredthat the concentration of cyclodextrin in the reaction mixture affectsthe conversion rate of the substrate into the products as well as theratio between the different products of the reaction, as shown in thetable below. shows the effect of cyclodextrin concentration in thereaction mixture on CBDA synthase reaction at pH 4.85.

As shown in the table below, increasing the concentration ofcyclodextrin from 0 mg/ml to 28 mg/ml in the CBDA synthase enzymereaction increases the conversion rate of CBGA to CBDA and CBCA, withthe highest conversion rates seen when cyclodextrin concentrations were8 mg/ml and 12 mg/ml (20% higher conversion rate comparing to nocyclodextrin added to the reaction).

Addition of cyclodextrin also slightly changes the ratio of CBDA:CBCA atpH 5. The highest CBDA:CBCA ratio (CBDA:CBCA 1.41:1) was observed whencyclodextrin concentration was 20 mg/ml and the lowest CBDA:CBCA ratio(CBDA:CBCA 1.04:1) was observed when cyclodextrin concentration was 16mg/ml.

TABLE 2 Effect of Cyclodextrin on CBDA Synthase Reaction Conversion Rateand Product Ratio CBDA:CBCA Cyclodextrin concentration Conversion rateratio FIG.  0 mg/ml 40% 1.13:1 11  2 mg/ml 57% 1.24:1 12  4 mg/ml N/AN/A N/A  8 mg/ml 61% 1.28:1 13 12 mg/ml 60% 1.33:1 14 16 mg/ml 50%1.04:1 15 20 mg/ml 53% 1.41:1 16 28 mg/ml 45% 1.24:1 17

The cyclodextrin may be α-cyclodextrin, β-cyclodextrin orγ-cyclodextrin. In some embodiments, the cyclodextrin issulfobuthylether β-cyclodextrin sodium salt or randomly methylatedβ-cyclodextrin. When present in the reaction mixture, the cyclodextrinis in a concentration of from about 0.001 to about 30 mg/ml. Preferably,the concentration of cyclodextrin in the reaction mixture is between 2and 28 mg/ml. In a most preferred embodiment, the concentration ofcyclodextrin in the reaction mixture is 8 mg/ml.

As shown in the table below and in FIGS. 11-17, with no cyclodextrin,increasing the amount of DMSO from 10% to 20% increased the conversionof CBGA from 13.5% to 45.8% overnight, and changed the ratio ofTHCA:CBCA from 3.33:1 to 2.24:1. Including cyclodextrin in the reactionwith 10% DMSO, increased the conversion of CBGA to 89.7% and gave aratio of 4.2:1 THCA:CBCA. Increasing the concentration of cyclodextrinto 40% or 60% gave the same results.

FIG. Condition CBGA THCA CBCA THCA:CBCA ID 10% DMSO, no 86.51% 10.40%3.0900%  3.37:1 1 cyclodextrin 20% DMSO, no 54.18% 32.39% 13.43% 2.41:12 cyclodextrin 10% DMSO, 20% 10.33% 72.37% 17.300%  4.20:1 3cyclodextrin 10% DMSO, 40% 11.50% 72.08% 16.42% 4.39:1 4 cyclodextrin10% DMSO, 60% 10.36% 73.98% 15.65% 4.73:1 5 cyclodextrin

The cannabinoids or cannabinoid analogs produced according to themethods of the invention aremay be single enantiomers with anenantiomeric purity of at least 95%, and preferably of at least 99%.

The inventors of the present application have also unexpectedlydiscovered that the pH of the reaction mixture affects the ratio betweenthe different cannabinoid products obtained. Accordingly, in a preferredembodiment, the pH of the reaction mixture is modified to obtain thecannabinoid products and/or cannabinoid analog products in the desiredratio.

Thus, when reacted with a compound of Formula I according to theinvention, THCA synthase may produce tetrahydrocannabinol (THCA),cannabichromene (CBCA), THCA and CBCA, or analogs thereof in differentratios, according to the pH of the reaction. Preferably, the reaction isperformed at a pH in a range between 3.8 and 7.2, and the methodproduces THCA, CBCA, or THCA and CBCA in a ratio as shown in thefollowing table at each specified pH:

TABLE 3 Effect of pH on THCA Synthase Reaction Products pH THCA CBCA 4 10 5 2.33 1 6 1 5.67 7 0 1

In summary, changing the pH of the THCA synthase enzyme reaction affectsthe products. At pH 4 THCA is the only product. At pH 5 the ratio ofTHCA:CBCA is 2.33:1. At pH 6 the ratio is reversed and the product mixis THCA:CBCA 1:5.67. At pH 7 CBCA is the only product. Under theseconditions, 98% of the compound of Formula I is converted into one ormore cannabinoids or cannabinoid analogs within two hours.

Similarly, when reacted wit a compound of Formula I, CBDA synthase mayproduce cannabidiol (CBDA), cannabichromene acid (CBCA), CBDA and CBCA,or analogs thereof in different ratios, according to the pH of thereaction. Preferably, the reaction is performed at a pH in a rangebetween 3.8 and 7.2, and the method produces CBDA, CBCA, or CBDA andCBCA in a ratio as shown in the following table at each specified pH:

TABLE 4 Effect of pH on CBDA Synthase Reaction Products pH CBDA CBCA 4.22.5 1 5 1.13 1 5.2 1 1.17 5.4 1 2.45 5.8 1 6.14 6.2 1 28.13 6.8 0 0

In summary, changing the pH of the CBDA synthase enzyme reaction affectsthe products. At pH 4.2 the CBDA:CBCA ratio is 2.5:1. At pH 5 the ratioof CBDA:CBCA is 1.13:1. At pH 6.8 there is no product forming from CBDAsynthase enzyme reaction. Under these conditions, 70% of the compound ofFormula I is converted into one or more cannabinoids or cannabinoidanalogs within two hours.

The invention also provides a method of producing one or morecannabinoids or cannabinoid analogs according to Formula II

wherein the method comprises the steps of: (a) reacting a compoundaccording to Formula III with a compound according to Formula IV;

in the presence of an enzyme that catalyzes the reaction of the FormulaIII and Formula IV compounds to form a Formula II compound; (b) reactingthe compound of Formula II with a cannabinoid acid synthase in areaction mixture comprising a solvent and an amphiphilic compound asdescribed above to produce one or more cannabinoids or cannabinoidanalogs; (c) isolating from the reaction mixture one or morecannabinoids or cannabinoid analogs produced in step (b); and (e)optionally decarboxylating the one or more cannabinoids or cannabinoidanalogs isolated in step (c). R in Formula III may be selected from —OH,halogen, —SH, or a —NR_(a)R_(b) group; R₁ and R₂ are each independentlyselected from the group consisting of —H, —C(O)R_(a), —OR_(a), anoptionally substituted linear or branched (C₁-C₁₀)alkylene, anoptionally substituted linear or branched (C₂-C₁₀)alkenylene, anoptionally substituted linear or branched (C₂-C₁₀)alkynylene, anoptionally substituted C₃-C₁₀ aryl, an optionally substituted C₃-C₁₀cycloalkyl, (C₃-C₁₀)aryl-(C₁-C₁₀)alkylene,(C₃-C₁₀)aryl-(C₂-C₁₀)alkenylene, and (C₃-C₁₀)aryl-(C₁-C₁₀)alkynylene, orR₁ and R₂ together with the carbon atoms to which they are bonded form aC₅-C₁₀ cyclic ring; R₃ is selected from the group consisting of H,—C(O)R_(a) and C₁-C₁₀ linear or branched alkyl. R₅ in Formula IV may beselected from the group consisting of a linear or branched(C₁-C₁₀)alkylene, a linear or branched (C₂-C₁₀)alkenylene, a linear orbranched (C₂-C₁₀)alkynylene, —C(O)— (C₁-C₁₀)alkylene,—C(O)—(C₂-C₁₀)alkenylene and —C(O)—(C₂-C₁₀)alkynylene; wherein anyalkylene, alkenylene, alkynylene, aryl, arylalkylene, or cycloalkylgroup is further substituted with one or more groups selected from thegroup consisting of —OH, halogen, —NR_(b)R_(c), —C(O)R_(a),—C(O)NR_(b)R_(c), (C₁-C₁₀)alkyl, —CN, (C₁-C₄)alkoxy, (C₁-C₄)haloalkyl,and (C₁-C₄)hydroxyalkyl; and R_(a), R_(b) and R_(c) are eachindependently —H, —OH, —SH, —NH₂, (C₁-C₁₀) linear or branched alkyl, ora C₃-C₁₀ cycloalkyl.

In one embodiment, R₅ is a (C₂-C₁₀)alkenylene selected from the groupconsisting of

and R₄ is a linear alkylene selected from the group consisting of CH₃,C₂H₅, C₃H₇, C₄H₉, C₅H₁₁, C₆H₁₃, C₇H₁₅ and C₈H₁₇. In a preferred aspectof the invention, R₅ is

and R₆ is selected from (C₁-C₁₀)alkylene, (C₂-C₁₀)alkenylene, —OH, —SH,NO₂, F, Cl, Br, —NH₂, or —NHR_(a).

In yet another embodiment, the invention provides a method for producinga tetrahydrocannabinol, cannabichromene, or both tetrahydrocannabinoland cannabichromene, or their analogs, wherein the method comprises thesteps of: (a) selecting a compound according to Formula I;

(b) reacting the compound of Formula I with a tetrahydrocannabinolicacid (THCA) synthase in a reaction mixture comprising a solvent and anamphiphilic compound as described above; (c) modifying at least oneproperty of the reaction mixture, such as the pH of the reaction, thenature and/or concentration of the non-aqueous solvent and/or theconcentration of an amphiphilic compound, such as cyclodextrin, toobtain a tetrahydrocannabinol, a cannabichromene, or bothtetrahydrocannabinol and cannabichromene, or their analogs as productsas described above; (d) isolating tetrahydrocannabinol, cannabichromene,or both tetrahydrocannabinol and cannabichromene, or their analogs fromthe reaction mixture; and (e) decarboxylating the tetrahydrocannabinol,cannabichromene, or both tetrahydrocannabinol and cannabichromene, ortheir analogs. R in Formula I may be selected from —OH, halogen, —SH, ora —NR_(a)R_(b) group; R₁ and R₂ are each independently selected from thegroup consisting of —H, —C(O)R_(a), —OR_(a), an optionally substitutedC₁-C₁₀ linear or branched alkylene, an optionally substituted C₂-C₁₀linear or branched alkenylene, an optionally substituted C₂-C₁₀ linearor branched alkynylene, an optionally substituted C₃-C₁₀ aryl, anoptionally substituted C₃-C₁₀ cycloalkyl, (C₃-C₁₀)aryl-(C₁-C₁₀)alkylene,(C₃-C₁₀)aryl-(C₂-C₁₀)alkenylene, and (C₃-C₁₀)aryl-(C₁-C₁₀)alkynylene, orR₁ and R₂ together with the carbon atoms to which they are bonded form aC₅-C₁₀ cyclic ring; R₃ is selected from the group consisting of H,—C(O)R_(a) and C₁-C₁₀ linear or branched alkyl; and R_(a) and R_(b) areeach independently —H, —OH, —SH, —NH₂, (C₁-C₁₀) linear or branchedalkyl, or a C₃-C₁₀ cycloalkyl.

In a different embodiment, the invention also provides a method forproducing a cannabidiol, cannabichromene, or both cannabidiol andcannabichromene, or their analogs comprising the steps of: (a) selectinga compound according to Formula I;

(b) reacting the compound of Formula I with a cannabinodiolic acid(CBDA) synthase in a reaction mixture comprising a solvent and anamphiphilic compound as described above; (c) modifying at least oneproperty of the reaction mixture, such as the pH of the reaction, thenature and/or concentration of the non-aqueous solvent and/or theconcentration of an amphiphilic compound, such as cyclodextrin, toobtain a cannabidiol, a cannabichromene, or both cannabidiol andcannabichromene, or their analogs as products; (d) isolatingcannabidiol, cannabichromene, or both cannabidiol and cannabichromene,or their analogs from the reaction mixture; and (e) decarboxylating thecannabidiol, cannabichromene, or both cannabidiol and cannabichromene,or their analogs. R in Formula I may be selected from —OH, halogen, —SH,or a —NR_(a)R_(b) group; R₁ and R₂ are each independently selected fromthe group consisting of —H, —C(O)R_(a), —OR_(a), an optionallysubstituted C₁-C₁₀ linear or branched alkylene, an optionallysubstituted C₂-C₁₀ linear or branched alkenylene, an optionallysubstituted C₂-C₁₀ linear or branched alkynylene, an optionallysubstituted C₃-C₁₀ aryl, an optionally substituted C₃-C₁₀ cycloalkyl,(C₃-C₁₀)aryl-(C₁-C₁₀)alkylene, (C₃-C₁₀)aryl-(C₂-C₁₀)alkenylene, and(C₃-C₁₀)aryl-(C₁-C₁₀)alkynylene, or R₁ and R₂ together with the carbonatoms to which they are bonded form a C₅-C₁₀ cyclic ring; R₃ is selectedfrom the group consisting of H, —C(O)R_(a) and C₁-C₁₀ linear or branchedalkyl; and R_(a) and R_(b) are each independently —H, —OH, —SH, —NH₂,(C₁-C₁₀) linear or branched alkyl, or a C₃-C₁₀ cycloalkyl. Thus, thepresent inventors have devised methods that produce differentcannabinoids and/or cannabinoid analogs in the desired ratio and in acontrolled manner, by varying the pH of the reaction, the nature and/orconcentration of the non-aqueous solvent and/or the concentration of anamphiphilic compound, such as cyclodextrin, in the reaction mixture.Apparatus and Methods for Producing Cannabinoids or Cannabinoid Analogs

An apparatus or system is provided for producing one or morecannabinoids or cannabinoid analogs according to the methods of theinvention. The apparatus may comprise a fermentor, a filter, abioreactor, and a control mechanism. FIG. 7 depicts an apparatus 100configured to produce at least one cannabinoid and/or at least onecannabinoid analog according to an embodiment. As shown in FIG. 7, theapparatus 100 includes a fermentor 10, a filter 20, a bioreactor 30, anda control mechanism (controller) 40. The fermentor 10 holds cell culturemedium 12 and a plurality of cells 14. The cells 14 produce and secretea cannabinoid acid synthase. The cells 14 grown in the fermentor 10 forthe manufacture of a cannabinoid acid synthase can be yeast, prokaryoticor eukaryotic cells that have been genetically modified to include anucleic acid sequence or a gene that encodes a cannabinoid acid synthaseprotein. In certain embodiments, the nucleic acid sequence that encodesa cannabinoid acid synthase protein is modified to include a yeast alphasecretion sequence at its 5′ end and to incorporate a 6-residuehistidine tag (SEQ ID NO: 9) at its 3′ end. The addition of the yeastalpha secretion sequence permits secretion of the cannabinoid acidsynthase protein into the medium 12 used for eukaryotic cell growth.Following production of cannabinoid acid synthase in the fermentor 10,the supernatant comprising the medium 12 and cells 14 (and cannabinoidsynthase), is transported along a pathway to the filter 20.

The filter 20 may filter the supernatant to at least partially separatethe cells 14 from the medium 12 containing the expressed enzyme.Typically, the filter 20 separates at least 80% of the total cells 14from the medium. In some embodiments, the filter 20 separates at least85%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, at least99%, or 100% of the total cells 14 from the medium 12. Followingfiltration, the cells 14 are transported back to the fermentor 10. Inone embodiment, the filter 20 can be a filtration and purificationsystem that includes multiple filters and reservoirs to purify thecannabinoid synthase.

After passing through the filter 20, the cannabinoid acid synthase flowsinto the bioreactor 30 and enters the bioreactor 30 through an inlet 32.The bioreactor 30 also includes an inlet 34 for reactants, such as thesubstrate CBGA or other substrates according to the Formula I compounddescribed above.

In some embodiments, the bioreactor 30 can be a column bioreactor havinga support 36. The support 36 may be a solid support that is impregnatedwith divalent metal ions or a support whose surface is functionalizedwith divalent metal ions. Typically, sepharose, agarose or otherbiopolymers are used as supports for binding divalent metal ions such asnickel, cobalt, magnesium and manganese. Such supports have a strongaffinity for the histidine tag that is present on the expressedcannabinoid acid synthase and can be used to sequester the synthase andseparate it from other non-essential proteins and debris that mayinterfere or impede cannabinoid synthesis.

The bioreactor 30 used for synthesizing cannabinoids is configured forbatch and continuous synthetic processes to permit commercial productionof pharmaceutically useful cannabinoids. In one embodiment, thebioreactor 30 is configured for batch synthesis in which the compositionof the medium, concentration of the enzyme and substrate are fixed atthe beginning of the process and not allowed to change during catalysis.Synthesis is terminated when the concentration of the desired product inthe medium of the bioreactor 30 reaches a predetermined value or theconcentration of substrate falls below a predetermined level, such as toa level where there is no detectable catalytic conversion of substrateto product.

In one embodiment, therefore, the His-tagged cannabinoid acid synthaseis sequestered onto a nickel containing resin support within thebioreactor 30 prior to the introduction of a known amount of substrate,for example, cannabigerolic acid (CBGA), or a compound of Formula I orFormula II into the bioreactor 30. In an alternate embodiment, CBGA or acompound of Formula I or Formula II can be present within the bioreactor30 having a nickel resin support prior to the introduction of the mediumcontaining a cannabinoid acid synthase into the bioreactor 30.

The progress of the reaction within the bioreactor 30 can be monitoredperiodically or continuously. For instance, an optical monitoring system50 may be utilized to detect the concentration of product in the mediumwithin the bioreactor as a function of time. Alternatively, the decreasein the concentration of substrate can be monitored to signal terminationof synthesis. The cannabinoid product thus produced can be readilyrecovered from the medium using standard solvent extraction orchromatographic purification methods. The monitoring system 50 may bepart of or may interact with a control mechanism 40 (a controller)described further below.

An alternative to the batch process mode is the continuous process modein which a defined amount of substrate and medium are continuously addedto the bioreactor 30 while an equal amount of medium containing thecannabinoid product is simultaneously removed from the bioreactor 30 tomaintain a constant rate for formation of product. The medium can enterthe bioreactor 30 through the inlet 32 and exit the bioreactor throughan outlet 38.

The conditions of the bioreactor can be controlled using a controlmechanism 40. The control mechanism 40 may be coupled to the bioreactor30 or, alternatively, may interact with the bioreactor 30 wirelessly orremotely. The control mechanism 40 may also be used to control theconditions of the fermentor 10, such the oxygen level, agitation, pH,and feed rate. The control mechanism 40 may also control the flow ofmaterials (e.g. by controlling at least one pump) into and out of thefermentor 10, filter 20, and bioreactor 30. In some embodiments, thecontrol mechanism 40 is configured to control the conditions of at leastone of the fermentor 10, the filter 20 and the bioreactor 30 based oninformation obtained from the optical monitoring system 50.

The control mechanism 40 may include a processing circuit having aprocessor and memory device. The processor and memory are configured tocomplete or facilitate the various processes and functions described inthe present application, such as controlling the pH, temperature, andpressure of the bioreactor 30, or altering the flow rate of medium intoor out of the bioreactor 30. In some embodiments, for facilitating thecontrol of pH, temperature, pressure and flow rate, the controlmechanism 40 may be configured to communicate with at least one sensorin a sensor suite 60. The sensor suite 60 may include a pH sensor 62, atemperature sensor 64, and a pressure sensor 66. The control mechanism40 may include a proportional-integral-derivative (PID) controller forfeedback-based control. The control mechanism 40 may be furtherconfigured to regulate the flow rate of materials into and out of thefermentor 10, the filter 20 and the bioreactor 30 via pulse widthmodulation (PWM) techniques.

FIG. 10 depicts the control mechanism 40. The control mechanism 40includes a processor 43 coupled to a communication bus 48. The controlmechanism 40 further includes a main memory 42, such as a random accessmemory (RAM) or other dynamic storage device, coupled to the bus 48 forstoring information, and configured to store instructions to be executedby the processor 43. The main memory 42 is further configured to storetemporary variables and intermediate information during execution ofinstructions by the processor 43. The control mechanism 40 mayadditionally include a read only memory (ROM) 44 or other static storagedevice connected to the bus 48 for storing information and instructions.Additionally, a storage device 46, such as a solid state device,magnetic disk or optical disk, may be coupled to the bus 48 forpersistently storing information and instructions.

Furthermore, the control mechanism 40 may be coupled (via the bus 48) toa display 77, such as a liquid crystal display, or active matrixdisplay, for displaying information to a user. In some embodiments, aninput device 11, such as a keyboard, may also be coupled to the bus 48for communicating information, and to convey commands to the processor43. In some embodiments, the input device 11 has a touch screen display.

In some embodiments, the bioreactor 30 is not a column reactor. Instead,as shown in FIG. 8, the bioreactor 30 comprises a plurality ofmicrotiter plates and is provided in a system 200. The system 200, likethe system 100, includes a controller 40 configured to control thebioreactor 30. The controller 40 may control the environmentalconditions of the bioreactor 30 and the supply of materials to thebioreactor 30, and may also control operations performed on theplurality of microtiter plates.

In some embodiments, each of the microtiter plates of system 200 has 96wells. In other embodiments, at least one microtiter plate has 384wells, 1,536 wells, 3456 wells, or 9600 wells. In embodiments with96-well microtier plates, an enzyme reaction may take place in each ofthe 96 wells. The reaction in each well make take place in a volume of0.5 ml or in a volume exceeding 0.5 mL. The apparatus described aboveare configured to produce cannabinoid acids or cannabinoid acid analogs,specifically, THCA and CBCA or CBDA and CBCA, by implementing thetechniques described below in reference to FIG. 9. FIG. 9 illustrates anautomated method (900) for producing cannabinoids or cannabinoid analogsaccording to an embodiment. The method includes providing cannabinoidCBG, DMSO, and a cannabinoid biosynthetic enzyme in each of the wells(901). The cannabinoid biosynthetic enzyme may be, for example, THCAsynthase. The cannabinoid biosynthetic enzyme is produced by thefermentor 10 by growing yeast transformed with a gene encoding THCAsynthase, as described above. The cannabinoid CBG, on the other hand, ischemically synthesized. The cannabinoid CBG, the DMSO and cannabinoidbiosynthetic enzyme may be considered to be the ‘starting materials’introduced into the bioreactor to ultimately yield at least onecannabinoid or cannabinoid analog. The cannabinoid CBG, DMSO, andcannabinoid biosynthetic enzyme may be provided in each of the wells viaautomatic pipetting. In other words, an apparatus such as the systems100, 200 may comprise mechanized componentry that may be controlled, forexample, by the control mechanism 40, so as to deliver an appropriateamount of at least one of the cannabinoid CBG, DMSO, and cannabinoidbiosynthetic enzyme to each well of the microtiter plates. In someimplementations, operations shown in FIG. 9 may be performed iterativelyby virtue of such automation. For example, an automated dispensingsystem or automated delivery system may be configured as a supplymechanism and used to deliver at least one of CBG, DMSO and THCAsynthase and a solvent. In some implementations, the systems 100, 200may be configured with a programmable sample changer configured toautomate sample preparation and transfer. The programmable samplechanger may be, for example, the Gilson 223 Sample Changer produced byGilson, Inc. of Middleton, Wis., USA, and may be operable withperistaltic and/or syringe pumps.

The method further includes reacting the cannabinoid CBG and thecannabinoid biosynthetic enzyme such as THCA synthase in the DMSO oncethese materials are distributed in the wells (902). The method furtherincludes, in some implementations, determining a ratio of THCA to CBCAor a ratio of CBDA to CBCA to be produced by the reaction (903). In someimplementations, the control mechanism 40 determines a quantity of THCAand a quantity of CBCA to be produced, or a quantity of CBDA and aquantity of CBCA to be produced. Next, the method includes determiningwhether the pH of the reaction mixture requires adjusting in order toyield the predetermined ratio of THCA to CBCA or the predetermined ratioof CBDA to CBCA (904). Specifically, the pH may be adjusted by alteringthe composition of the reaction mixture to obtain a desired ratio ofTHCA:CBCA or a desired ratio of CBDA:CBCA. The reaction culminates inthe production of THCA and CBCA or CBDA and CBCA (905).

The method further includes automatically pipetting a solvent into eachwell of the microtiter plate (906). The addition of the solvent resultsin cessation of the reaction. The method additionally includes,following the introduction of the solvent into the wells and cessationof the reaction, recovering cannabinoids or cannabinoid analogs in thesolvent layer.

Once the reaction has ceased, the resulting solvent layer is removed(907), and the cannabinoids or cannabinoid analogs are recoverable. Morespecifically, the cannabinoids or cannabinoid analogs are recoverablefrom a solvent fraction present in each of the wells via vacuumevaporation or ethanol extraction (908). In some embodiments, a rotaryevaporator is used to remove the solvent. The rotary evaporator may bean automated rotary evaporator such as the fully automated POWERVAP®rotary evaporator produced by Genser Scientific Instruments ofRothenburg ob der Tauber, Germany. Upon removal of the solvent, thecannabinoids or cannabinoid analogs are left in the bottoms of thewells. The method further includes re-suspending the cannabinoids orcannabinoid analogs (909). The cannabinoids or cannabinoid analogs maybe re-suspended in ethanol, liposomes, or lipid micelles.

The process illustrated in FIG. 9 permits recovery of cannabinoids orcannabinoid analogs that may be readily formulated into pharmaceuticalsand marijuana-infused products including beverages, confectionery, andcosmetics, among other examples. The cannabinoids or cannabinoid analogsmay be readily purified via HPLC for pharmaceutical applications.

In at least one implementation, 0.5 mg of buffered CBG, buffered THCAsynthase or buffered CBDA synthase with or without stabilizer, and DMSOare automatically pipetted into each of a plurality of wells of amicrotiter plate. The DMSO that is added may have a final concentrationof 20%, in some implementations. The ensuing reaction in each of theplurality of wells generally yields approximately 0.5 mg of cannabinoidswhen incubated for 2 hours, 4 hours, 12 hours, and 24 hours. Thus, for amicrotiter plate including 96 wells, the system 100 produces about 48 mgof cannabinoids. It follows that the amount of cannabinoids produced‘scales up’ when a plurality of microtiter plates are used. For example,if 21 microtiter plates are used, each having 96 wells, then 1008 mg(about 1 gram of cannabinoids) may be produced according to thetechniques described above. Using 315 microtiter plates producesapproximately 150 grams of cannabinoids. In some implementations,volumes greater than 0.5 mg may be used for the reaction mixture.

In some embodiments, the processor of the controller can be implementedas a general purpose processor, an application specific integratedcircuit (ASIC), one or more field programmable gate arrays (FPGAs), agroup of processing components, or other suitable electronic processingcomponents. The memory device (e.g., memory, memory unit, storagedevice, etc.) is one or more devices (e.g., RAM, ROM, Flash memory, harddisk storage, etc.) for storing data and/or computer code for completingor facilitating the various processes and functions described above. Thememory device may be or include volatile memory or non-volatile memory.The memory device may include database components, object codecomponents, script components, or any other type of informationstructure for supporting the various activities and informationstructures described in the present application. According to oneembodiment, the memory device is communicably connected to the processorvia the processing circuit and includes computer code for executing(e.g., by the processing circuit and/or processor) one or more processesdescribed herein.

The present disclosure contemplates methods, apparatus and programproducts on any machine-readable media for accomplishing variousoperations, such as controlling the conditions of the bioreactor. Theembodiments of the present disclosure may be implemented using existingcomputer processors, or by a special purpose computer processor for anappropriate system, incorporated for this or another purpose, or by ahardwired system. Embodiments within the scope of the present disclosureinclude program products comprising machine-readable media for carryingor having machine-executable instructions or data structures storedthereon. Such machine-readable media can be any available media that canbe accessed by a general purpose or special purpose computer or othermachine with a processor. By way of example, such machine-readable mediacan comprise RAM, ROM, EPROM, EEPROM, CD-ROM or other optical diskstorage, magnetic disk storage, other magnetic storage devices, solidstate storage devices, or any other medium which can be used to carry orstore desired program code in the form of machine-executableinstructions or data structures and which can be accessed by a generalpurpose or special purpose computer or other machine with a processor.When information is transferred or provided over a network or anothercommunications connection (either hardwired, wireless, or a combinationof hardwired or wireless) to a machine, the machine properly views theconnection as a machine-readable medium. Thus, any such connection isproperly termed a machine-readable medium. Combinations of the above arealso included within the scope of machine-readable media.Machine-executable instructions include, for example, instructions anddata which cause a general purpose computer, special purpose computer,or special purpose processing machines to perform a certain function orgroup of functions.

The control mechanism may further include additional devices, such as akeyboard and display, to allow a user to interact with the controlmechanism to control the conditions of the bioreactor. For example, thedisplay may include a screen to allow a user to monitor changes in pH,temperature, pressure, and flow rate of the bioreactor, or to monitorany other condition of the system for producing cannabinoids orcannabinoid analogs. The present invention is further described by thefollowing examples which are not meant to limit the scope of the claims.

EXAMPLES

A. Molecular Cloning, Screening and Expression of Protein from HighYield Yeast Transformants

1. Restriction Digestion.

THCA α plasmid DNA and CBDA α plasmid DNA were linearized by digestingeach plasmid with Pme I or Spe I restriction enzymes at 37° C. for twohours. Linearized plasmids were verified on 0.8% agarose gel byelectrophoresis. Qiagen Gel Extraction kit was used to extract thelinearized plasmid from the agarose gel and the plasmids were frozen at−20° C. until use.

2. Preparation of Electrocompetent Yeast Cells.

Electrocompetent PichiaPink (pPink) cells were made by inoculating 10 mLof YPD media with a glycerol stock of a genetically engineered Ade2,pep4 knockout pPink yeast strain 2. These cells were grown overnight ina 125 ml baffled flask at 28° C., using a shaker spinning at 270 rpmuntil the OD₆₀₀ of the culture reached a value of 1.3 units indicatinglog phase growth. This culture was then added to 100 ml of YPD media andallowed to incubate overnight under the same conditions. The OD₆₀₀ waschecked hourly and after a 12 hour incubation period reached a value of1.3 units.

After reaching log phase growth the cells were transferred to a 500 mlcentrifuge tube and spun down for 5 minutes at 4° C. and 2500 rpm. TheYPD broth was decanted and 250 ml of sterile ice-cold water was addedand the cells re-suspended. The cells were then centrifuged at 4° C.,2500 rpm for another 5 minutes, re-suspended with an additional 250 mlof water to ensure removal of all YPD media and centrifuged under thesame conditions again. The water was then decanted and 50 ml of sterileice-cold water was added and the cells re-suspended and centrifugedunder the same conditions. The water was then decanted and 10 ml ofsterile, ice-cold 1M sorbitol was added and the cells re-suspended. Thesuspension was then transferred to a sterile 15 ml conical tube andcentrifuged under the same conditions as before. The 1M sorbitol wasthen decanted, 300 μl of sterile ice-cold 1M sorbitol was added and thecells were re-suspended and placed on ice for use.

3. Electroporation

The previously frozen linearized plasmid DNA was thawed on ice and 80 μlof the electrocompetent pPink cells were added to the tube. This volumewas then transferred to a 0.2 cm electroporation cuvette and incubatedon ice for 5 minutes. The cuvette was then pulsed at 1640 V, 200Ω, and25 μF for a total pulse time of approximately 4 minutes. Immediatelyafter pulsing, 1 ml of YPDS media was added to the cuvette and mixed bypipetting. The cuvette was then placed in a 28° C. incubator, withoutshaking, for 2 hours, after which 300 μl was spread onto fresh PADplates. The PAD plates were then placed into the 28° C. incubator forapproximately 7-10 days and inspected each day for cell growth.

4. Screening

White colonies are indicative of positive expression of the gene ofinterest, whereas red colonies indicate no expression. All whitecolonies were selected and re-streaked onto fresh PAD plates and allowedto grow for 3-5 days until individual colonies appeared. A single colonywas then used to inoculate 10 ml of BMGY in a 125 ml baffled flask andplaced into an incubator overnight shaking at 28° C. and 270 rpm. Whenthe OD₆₀₀ reached 1.2-1.5 (after 1:10 dilution in water) the inoculumwas transferred to a 50 ml conical tube and centrifuged at 2500 rpm for5 minutes. The BMGY was decanted and 1 ml of BMMY was added. The tubeswere then covered with air porous tape to allow for sterile air exchangeand placed into the shaking incubator at 28° C. and 270 rpm.

After 24 hours 100 μl of the sample were removed and 100 μl of 40%methanol were added. The removed portion was then centrifuged at 12,000rpm for 5 minutes and the supernatant and pellet were saved as T=1(day 1) samples. This procedure was then repeated after 48 hours (T=2).After 72 hours (T=3) the remaining sample was harvested as the finaltime point. The T=3 supernatant was then spun through an Amicon 30 kDprotein filter and run on an SDS-PAGE for visualization of protein.

5. Enzymatic Conversion

Samples that had greater than 20% conversion of CBGA to CBDA over 4-24hours were then scaled up. Briefly, enzymatic conversion reaction was asfollows: 25 μl of cell free supernatant from the T=3 samples wasincubated for 2 hours at 30° C., with 25 μl of a 1 mg/ml CBGA stock inDMSO in 200 μl of pH 4.8, 100 mM citrate buffer. Reaction yielded afinal concentration of CBGA of 0.1 mg/ml at pH 5.0.

For Scale-Up, a single colony was used to inoculate 10 ml of BMGY in a125 ml baffled flask which was incubated overnight at 28° C. and 270rpm. The OD₆₀₀ was measured after 24 hours, and once it reached 1.2 the10 ml suspension was then used to inoculate 90 ml of BMGY in a 1 Lbaffled flask. The suspension was then allowed to incubate overnight at28° C. and 270 rpm. When the OD₆₀₀ reached 1.2-1.5 the inoculum was thentransferred to a 500 ml centrifuge bottle and pelleted at 2500 rpm for 5minutes.

The BMGY was decanted and the cell pellet washed with 10 ml of BMMY.After 2 washings the pellet was re-suspended with 10 ml of BMMY,transferred to a 500 ml baffled flask and allowed to incubate overnightat 28° C. and 270 rpm. After 24 hours 1 ml of the sample was removed(T=1) and 1 ml of 40% methanol was added. This was repeated after 48 andafter 72 hours the full sample volume was harvested, separated andanalyzed.

Table 5 below shows the results of small scale screening samples withgreater than 20% conversion of CBGA to THCA that were selected forscale-up.

TABLE 5 Small Scale Screening Samples with Greater than 20% Conversionof CBGA to THCA % Conversion of CBGA to THCA in reaction Sample IDcontaining 0.1 mg/ml CBGA. Spe THC #3 20.6 Spe THC #4 28.7 Spe THC #2220.6 Spe THC #23 18.7 Pme THC #5 32.5 Pme THC(2) #1 29.1 Pme THC(2) #2A27.2 Pme THC(2) #25 31.6 Pme THC(2) #36 27.7 Pme THC(2) #41 32.5 PmeTHC(2) #42 27.6 Pme THC(2) #46 40.7 Pme THC(2) #51 26.8 Pme THC(3) #155.2 Pme THC(3) #11 35.0 Pme THC(3) #17 69.9 Pme THC(3) #19 36.8 PmeTHC(3) #20 34.36. Cloning Strategy for Generating Multi-Copy GOI Inserts In Vitro.

An alternate yeast expression system was used to obtain transformedcells having one or more copies of the gene of interest. The multi-copyPichia Expression Kit from Invitrogen was used to construct new plasmidsthat could generate multi-copy gene inserts in vitro or in vivo.

In Vitro Generation of Multi-Copy Inserts

To generate multi-copy GOI inserts in vitro, the pAO815 vector was usedto clone the gene of interest. α-CBDA synthase and α-THCA synthase werecut with EcoR I and Bam HI from pPink-HC plasmid by incubating 100 ng ofthe pPink-HC vector containing the α-CBDA synthase gene or the α-THCAsynthase gene with 1 μl of EcoR I buffer, 1 μl of each restrictionenzyme (10 units/μl) and 1 μl of BSA in 20 μl total reaction volume at37° C. for 2 hr. 100 ng of pAO815 vector was also digested with Eco R Iand Bam HI enzymes following the same protocol.

After digestion, the GOI and vectors mixture were run on a 0.8% agarosegel at 95 V for 1 hr. Bands of correct size were excised and extractedfrom the gel with Invitrogen gel extraction kit. The linearized vectorand gene inserts were ligated using T4 DNA ligase protocol from NEB®.Upon ligation, the circular vector containing the gene of interest wastransformed into E. coli Top 10 F⁻ cells to harvest plasmid byelectroporation at 1500 V, 200Ω and 25 μF for 4 milliseconds. Thetransformed cells were then mixed with 250 μl of SOC medium (providedwith One Shot® Top 10 Electrocomp™ E. coli from Invitrogen) and platedon a LB-Amp100 plate at 37° C. overnight. The next morning, positivecolonies were identified with colony PCR protocol with 5′ AOX1 and3′AOX1 primers. Positive colonies containing the gene of interest weregrown in liquid LB-Amp100 media overnight at 37° C. The next day plasmidmini-preps were done with Invitrogen's fast prep kit and theconcentration of the plasmid was analyzed on 0.8% agarose gel beforefurther amplification.

The recombinant pAO815 plasmid containing the alpha-THCA synthase andalpha-CBDA synthase genes was divided into 2 batches, one batch was usedas a vector in which was inserted a second copy of the gene of interestand one batch was used for extracting the alpha-THCA synthase oralpha-CBDA synthase genes. The vector batch was first digested with BamHI following NEB's single digest protocol. The second batch was digestedwith Bgl II and Bam HI restriction enzymes. The linearized vector andgenes were purified on a 0.8% agarose gel and extracted. The vector andgenes were then ligated following NEB's T4 DNA ligase protocol and thentransformed into E. coli Top10 F⁻ cells by electroporation as describedabove. The cells were incubated at 37° C. overnight and then screenedfor the correct gene insert by PCR. Gene sequences were confirmed bysequencing. The multi-copy plasmids were linearized at the His4 sequenceregion by restriction enzyme digestion and transformed into competentPichia pastoris strain G115 (his4, Mut+) cells. The transformed cellswere grown on His⁻ plates for screening. Screening was done on His⁻plates to confirm integration of the plasmid at the His site of thePichia Pastoris genome. Positive colonies were chosen for methanolinduction of protein, time points protein SDS-gel and enzyme assay.

In Vivo Generation of Multi-Copy Inserts

To generate multi-copy GOI inserts in vivo, the pPIC-3.5K vector wasused as the backbone to carry and insert one or more copies of theα-CBDA synthase gene or the α-THCA synthase gene into the Pichiapastoris GS115 strain genome. α-CBDA synthase and α-THCA synthase geneswere excised out with Pme I and Bam HI from pPink-HC plasmid, separatedfrom the pPink-HC backbone on a 0.8% agarose gel at 95 V for 1 hr andextracted from the gel with Qiagen or Invitrogen gel extraction kit.pPIC-3.5K plasmid was digested by PmeI and BamHi from NEB, run on 0.8%agarose gel and extracted from the gel with Qiagen or Invitrogen gelextraction kit.

The linearized vector and gene inserts were ligated together usingInvitrogen T4 DNA ligase protocol from NEB®. Ligated circularrecombinant plasmids were electroporated into E. coli Top 10 F⁻ strainand the cells were plated on LB-Amp-100 plates. The plates wereincubated were incubated overnight at 37° C. for colonies to form.Colony PCR was applied to verify successful transformation and coloniesbearing pPIC-3.5K-alpha-THCA synthase or pPIC-3.5K-alpha-CBDA synthasewere re-streaked on new LB-Amp-100 plates to generate more plasmids.

pPIC-3.5K-alpha-THCA synthase and pPIC-3.5K-alpha-CBDA synthase wereinserted into GS115 strain by electroporation as described above. Thetransformed GS115 cells were then plated on YPD-geneticin plates with0.25 mg/mml-3 mg/ml geneticin to select for one or more THCA synthasegene and CBDA synthase gene copy colonies. Colonies grown on 3 mg/mlYPD-geneticin plates were selected for THCA synthase and CBDA synthaseproduction screening.

Results

The conversion rate from CBGA to THCA and CBCA was greater than 90% intwo hours using crude fermentation supernatant (FIGS. 3 and 5).

The conversion rate from CBGA to CBDA and CBCA was greater than 70%overnight using crude fermentation supernantant. (FIG. 6).

7. Enzyme Purification

The cannabinoid acid synthase enzymes thus obtained were purified bysize exclusion chromatography (SEC) using a 2.2 cm inner diameter columnand 5 ml supernatant in a column volume to crude enzyme supernatantratio of 20:1. Briefly, 10 g of dry sephadex beads were measured andadded to a Pyrex glass container. 100 ml of 50 mM Phosphate buffer pH7.4 were added to Bio-GEL P-100 beads with excess amount and let sit formore than 12 hours. (P-100 beads swollen 12× when completely hydrated).Using a vacuum pump, the hydrated P-100 beads and another 1 L pH 7.4 50mM Phosphate buffer were de-gassed to cause the beads to settle in theexcess buffer. The buffer was poured off, and 100 mL de-gassed Phosphatebuffer were poured into the beads, such that the beads settled again.These steps were repeated two more times. The hydrated P-100 was thenpoured into a glass column until 2-5 cm of the gel bed was formed, thenmore gel was poured to the desired height and let it settle. The columnthus formed was stored at 4° C. 5 mL of either THCA or CBDA synthasecrude supernatant was run through the column at 4° C. and the fractionswere collected at 5 mL/fraction for 25 fractions. All fractions weresaved, stored at 4° C. and analyzed for enzyme activity and by SDS-PAGEgel to examine purification efficiency and resolution.

B. Cannabinoid and Cannabinoid Analog Enzymatic Production

1. Enzymatic Assay Conditions

The Standard CBDA synthase enzyme/THCA synthase enzyme reaction assayconditions were as follows: enzyme reaction was conducted in a 1.5 mlEppendorf snap cap tube. 25 μl substrate, such as CBGA, dissolved inDMSO at 1.0 mg/ml in 200 μl of 100 mM citrate buffer pH 4.85 wasincubated with 25 μl enzyme solution at 30° C. for 2 hours. The reactionwas terminated by the addition of 250 μl MeOH and analyzed by HPLC.

Enzyme activity was tested under a variety of conditions as follows:

1. Different solvents and conditions were tested to enhance substratesolubility and delivery, including but not limited to DMSO, DMF, IPA,cyclodextrin (CD), SDS, Triton-X.

2. Assays were run at pH's 4, 5, 6, 7, and 8.

3. Enzyme assays were run in either Sodium phosphate buffer or Citratebuffer with or without SDS or Triton-X

4. Enzyme assays were run under a variety of ionic strengths

5. Results of incubation times between 2 hrs to 4 days were compared.

Results

Table 6 below shows that DMSO, DMF, IPA and cyclodextrin facilitatedsolubilization of cannabinoids. Cyclodextrin solubilized up to 20-25 g/Lof CBGA for conversion. Enzymatic rate was enhanced when 20% DMSO (v/v)was added to the reaction mixture and THCA synthase produced both THCAand CBCA in the reaction (Table 7).

TABLE 6 Effects of solvents on THCA Synthase Activity Reaction Condition% CBGA Studies Parameters conversion THCA:CBCA 100 mM 100 mM Cit 50 ugEnzyme in 84 1.066:1  Solvents DMF 100 mM Cit 50 ug Enzyme in 85 7.96:1DMSO 100 mM Cit 50 ug Enzyme in 81 12.34:1  CD 400 mM cit 50 ug Enzymein 61 11.9:1 IPA 100 mM NaP 50 ug Enzyme in 79 1.11:1 20 ul CD 100 mMNaP 50 ug Enzyme in 72 1.22:1 20 ul SDS 100 mM Cit 50 ug Enzyme + 810.45:1  SDS in CD

TABLE 7 Effects of DMSO Concentration on THCA Synthase Rate and ProductRatio DMSO FASTER THCA:CBCA  0%  1 X 10% 1.2 X 10:1  20% 2.5 X 5:1 25% —1:1 30% 0.3 X

The effect of pH on THCA Synthase activity is shown in Tables 8 and 9below.

TABLE 8 Effects of pH on THCA Synthase Activity pH THCA CBCA 4 1 0 52.33 1 6 1 5.67 7 0 1

In summary, changing the pH of the THCA synthase enzyme reaction affectsthe products. At pH 4 THCA is the only product. At pH 5 the ratio ofTHCA:CBCA is 2.33:1. At pH 6 the ratio is reversed and the product mixis THCA:CBCA 1:5.67. At pH 7 CBCA is the only product.

TABLE 9 Effects of pH and Cyclodextrin on THCA Synthase ActivityReaction % CBGA THCA Condition Parameters conversion CBCA pH 400 mM CitpH 5.0, 50 μg Enzyme 59 14.9:1 exchange in CD 400 mM Cit pH 6.5, 50 μgEnzyme 42  1.1:1 in CD 400 mM NaPi pH 5.0, 50 μg Enzyme 59 17.37:1  inCD 400 mM NaPi pH 6.5 50 μg enzyme 65 1.11:1 20 μg in CD

The effect of pH on CBDA synthase activity is shown in Table 10 below.

TABLE 10 Effects of pH on CBDA Synthase Activity pH CBDA CBCA 4.2 2.5 15 1.13 1 5.2 1 1.17 5.4 1 2.45 5.8 1 6.14 6.2 1 28.13 6.8 0 0

In summary, changing the pH of the CBDA synthase enzyme reaction affectsthe products. At pH 4.2 CBDA:CBCA ratio is 2.5:1. At pH 5 the ratio ofCBDA:CBCA is 1.13:1. At pH 6.8 there is no product forming from CBDAsynthase enzyme reaction.

These results clearly show that it is possible to control the ratio ofTHCA:CBCA produced by the THCA synthase by controlling the pH of theenzymatic reaction. Enzyme assays were run in either Sodium phosphatebuffer or Citrate buffer with or without SDS or Triton-X.

The effect of different concentrations of cyclodextrin on cannabinoidacid synthase activity was evaluated. The results for the CBDA synthaseat pH 4.85 are shown in Table 11 below.

TABLE 10 Effect of Cyclodextrin on CBDA Synthase Reaction ConversionRate and Product Ratio Cyclodextrin concentration Conversion rateCBDA:CBCA ratio  0 mg/ml 40% 1.13:1  2 mg/ml 57% 1.24:1  4 mg/ml N/A N/A 8 mg/ml 61% 1.27:1 12 mg/ml 60% 1.33:1 16 mg/ml 50% 1.04:1 20 mg/ml 53% 1.0:1 28 mg/ml 45% 1.24:1

These results clearly show that the concentration of cyclodextrin in thereaction mixture affects the enzymatic conversion rate of the substrateinto the products as well as the ratio between the different products ofthe reaction.

These experiments also showed that the optimal cyclodextrin (CD):CBGAratio in the enzyme reaction mix was 11:1 (mass:mass) or 4:1 (molarratio) for CBDA synthase, and that the optimal cyclodextrin (CD):CBGAratio in the enzyme reaction mix was 28:1 (mass:mass) or 7.3:1 (molarratio) for THCA synthase CD:CBGA. The presence of cyclodextrin in thereaction mix in such concentration resulted in 98% conversion in 2 hours(data not shown).

B. Cannabinoid Extraction and Purification

Cannabinoids and cannabinoid analogs obtained from the enzymaticreactions with the cannabinoid acid synthase as described above wereextracted by solvent extraction as follows:

Solvent was added to the reaction mix at a ratio of 1:3 (v/v), themixture was vigorously vortexed at room temperature for 2 minutes andcentrifuged at 3200 g for 10 minutes. The solvent fraction was separatedand stored in a glass vial. These steps were repeated and allextractions were combined and analyzed by HPLC.

C. Production of Cannabinoid Acid Synthase Enzymes by Fermentation

Cannabinoid acid synthase enzymes were produced by fermentationfollowing Invitrogen ‘Pichia Fermentation Process Guidelines’. Somemodifications were as follows:

A. Inoculum Flask Preparation

From a frozen glycerol stock of Pichia strain GS115 (Mut+, Arg+, His−),a YPD plate was inoculated. After 48 hours a single colony on YPD wasused to inoculate 300 ml of BMGY, in a 2 L baffled flask. The culturewas grown at 28° C., 270 rpm, until OD₆₀₀ reached 2-6 (approximately 15hours).

B. Fermentor Preparation/Batch Glycerol

After sterilization and cooling of the 3.5 L of Basal Salts Medium inthe Glass vessel of the BioFlo 3000 Fermentor, the temperature was setto no less than 27° C. and no more than 30° C. Aeration and Agitationwere set to the PID mode (dissolved Oxygen dependent). pH wascontinually adjusted to 6.5 with 30% NH4OH. The Fermentor was inoculatedwith 300 ml of the culture generated above. 200 ml of 20% casamino acidsprepared in sterile 100 mM, pH 6.5 Phosphate Buffer, were added. Thedissolved oxygen was adjusted to be maintained above 20%. After theglycerol from the BMGY medium was completely consumed (approximately 24hours), a 10 ml sample was taken at the end of this first fermentationstage and analyzed for cell growth (OD600) and wet cell weight. Thepellet was frozen at −80° C. for later analysis of protein. The samplingwas repeated at end of each stage.

C. Glycerol Fed-Batch Phase

50% w/v glycerol with 12 ml PTM trace salts per liter of glycerolsolution was added to increase cell biomass. Feed rate was set to 18.15ml/hr./liter initial fermentation volume. Glycerol feed was continueduntil wet cell weight reached 180-220 g/liter and DO spike was used tomonitor the end point of glycerol fed batch phase.

D. Methanol Fed-Batch Phase

Methanol induction was initiated after all glycerol was consumed toinduce the AOX1 promoter and express the cannabinoid synthases. 100%methanol with 12 ml PTM trace salts per liter of methanol was added.Feed rate was initially set at 3.6 ml/hr./liter initial fermentationvolume. Agitation, aeration and oxygen feed were adjusted for the nexttwo hours to maintain the DO above 20%. A steady DO reading inferred afull adaptation to methanol at which point methanol feed was doubled to7.3 ml/hr./liter. After 2 hours methanol feed was further increased to10.9 ml/hr./liter initial fermentation volume. After about 2 hours or atthe first sign of foaming, 20-50 μl Sterile Pure Anti-Foam 204, Sigmawere added so as to keep the headspace of the fermentor clear andprevent the foam from interfering with the agitation and various feeds.Additional 20-50 μl aliquots were added as needed approximately once aday or every other day of the entire run of fermentation. Once 10.9ml/hr./liter was established, enzyme activity was measured and monitoredevery 8 hours thereafter. Fermentation was stopped 5 days after initialinoculation or upon reaching a plateau in protein concentration.

E. Harvesting Cells and Supernatant

At harvest time, the final fermentation volume was almost double theinitial volume. The cell density was increased to ˜400 g/liter wetcells. The 7 liter of culture was collected into 500 ml centrifugebottles and centrifuged at 10,000 RPM for 15 min to separate cells fromthe supernatant. The supernatant was concentrated 10× using TangentialFlow Filtration. A sample of supernatant was loaded onto apolyacrylamide gel for protein analysis. THCA synthase was around 80KDa. 30 Kda TFF filter was used to concentrate the fermentationsupernatant 10×. A portion of the TFF concentrated supernatant wasloaded onto a nickel column for purification of the enzyme. A portion ofthe original fermentation supernatant was fractioned by ammonium sulfateprecipitation (45%-75%).

F. Standard Enzyme Activity Assay

In 200 μl of 100 mM pH 4.8 Citrate buffer; 25 μl Substrate (CBGA)dissolved in DMSO at 1 mg/mL concentration; and 25 μl enzyme(supernatant) were added in a 1.5 mL Eppendorf snap cap tube. The tubewas incubated at 30° C. for 2 hours and the reaction was terminated byadding 250 μl MeOH. Activity of the enzymes was analyzed by HPLC.

E. Concentration/Purification of Cannabinoid acid synthase Enzymes fromFermentation

After fermentation the cells were separated from the supernatant bycentrifugation at 10,000 RPM×15 min. The enzyme was then concentratedand purified as follows: the supernatant was concentrated 10× usingTangential Flow Filtration and then fractionated using ammonium sulfateprecipitation; the protein fraction salting out between 45%-75%(NH4)2SO4 contained the synthase. The TFF filtered supernatant wasloaded onto a nickel column for purification of the enzyme.

F. Chemical Synthesis of Cannabinoid Substrates

A. Synthesis of Geraniol (3,7-Dimethylocta-2,6-dien-1-ol,)

Geraniol was obtained by distillation of palmarosa oil. Palmarosa oil(New Directions Aromatics) was distilled under reduced pressure and thefractions that distil between 139-145° C. and under a reduced pressureof 25 mm Hg were pooled to obtain pure geraniol.

B. Synthesis of Olivetol

Olivetol was synthesized using a published procedure (Focella, A, etal., J Org. Chem., Vol. 42, No. 21, (1977), p. 3456-3457).

1. Methyl 6-N-Pentyl-2-hydroxy-4-oxo-cyclohex-2-ene-1-carboxylate

To a stirring solution of sodium methoxide (32.4 g, 0.60 mol) anddimethyl malonate (90 g, 0.68 mol) in 230 mL of anhydrous methanol wasadded portion wise 75 g (0.48 mol) of 90% 3-nonen-2-one. The reactionmixture was then refluxed for 3 h under N₂ and allowed to cool to roomtemperature. The solvent was distilled under reduced pressure and theresidue dissolved in 350 mL of water. The slurry of white crystals andthe almost clear solution was extracted thrice with 80 mL of chloroform.The aqueous layer was acidified to pH 4 with concentrated HCl and thewhite precipitate that formed was allowed to stand overnight prior tofiltration. The crystals were dried at 50° C. under high vacuum for 5hours to yield 106.5 g (0.4416 mol) (92%) of methyl6-n-Pentyl-2-hydroxy-4-oxo-cyclohex-2-ene-1-carboxylate (mp 96-98 C).The product was recrystallized using a mixture of petroleum ether:ethylacetate (9:1), and gave 94 g of pure methyl6-n-Pentyl-2-hydroxy-4-oxo-cyclohex-2-ene-1-carboxylate (melting pointof 98-100 C).

2. 1-N-Pentyl-3, 5-dihydroxybenzene (Olivetol)

To a stirring ice-cooled solution of methyl6-N-pentyl-2-hydroxy-4-oxo-cyclohex-2-ene-1-carboxylate (58.4 g, 0.24mol) dissolved in 115 mL dimethylformamide was added dropwise 37.9 g(0.23 mol) of bromine dissolved in 60 mL of dimethylformamide. At theend of the addition (ca. 90 min) the reaction mixture was slowly heatedto 80° C. during which time the evolution of carbon dioxide became quitevigorous.

The reaction was maintained at this temperature until gas evolution hadceased following which the reaction was further heated to 160° C. andheld at this temperature for approximately 10 hours. After heating, thereaction was allowed to cool and the solvent DMF was removed underreduced pressure. The residue thus obtained was treated with water (80mL) and extracted twice with 250 mL of ether. The combined ether layerswere washed with water, then washed with 2×80 mL of a 10% solution ofsodium bisulfite, 2×80 mL of a 10% solution of acetic acid, and thenagain with water.

After drying over anhydrous sodium sulfate the solvent was removed underreduced pressure to give 46.8 g of a viscous oil. The oil was distilledunder reduced pressure to give 30.3 g (0.168 mol) (69.3%) of olivetol asproduct. HPLC analysis indicated 97.5% purity.

C. Synthesis of CBG

CBG was synthesized following the protocol disclosed by Taura et al.,(1996), The Journal of Biological Chemistry, Vol. 271, No. 21, p.17411-17416.

1. Synthesis of 2-[(2E)-3, 7-dimethylocta-2,6-dienyl]-5-pentyl-benzene-1,3-diol (Cannabigerol (CBG))

Geraniol (3 g, 0.0194 mol) and olivetol (2 g, 0.0111 mol) were dissolvedin 400 mL of chloroform containing 80 mg of p-toluenesulfonic acid ascatalyst and the reaction mixture was stirred at room temperature for 12h in the dark. After 12 hours, the reaction mixture was washed withsaturated sodium bicarbonate (400 mL) and then with H₂O (400 mL). Thechloroform layer was concentrated at 40 C under reduced pressure, andthe residue obtained was chromatographed on a 2.0 cm×25 cm silica gelcolumn using benzene (1000 mL) as the eluent to give 1.4 g (0.00442mol)(39.9%) CBG as product.

Alternatively crude CBG was purified as follows. To a 250 mL beaker wasadded 7.25 g crude CBG and 50 mL benzene. The flask was swirled todissolve the CBG and 50 g silica gel was added, along with a stir bar.The solution was stirred overnight, and then poured into a 44 cm×2.75 cmcolumn. The column was eluted with 300 mL benzene. The eluent,approximately 70 mL fractions were assayed for CBG. Fractions 1, 2, and3 (˜230 mL) that contained CBG were combined and the solvent removedunder pressure to give 6.464 g residue containing >80% CBG, having apurity suitable for use in the next synthetic step.

In one embodiment, crude CBG was purified by mixing 7.25 g crude CBGresidue with a slurry of silica gel (50 mL), in a 250 ml Beaker. Thismixture was slowly agitated for 1 hour and then vacuum filtered using afine mesh filter paper. The filter cake was washed with 250 ml benzeneuntil a clear filtrate was obtained. The solvent from the filtrate wasremoved under reduced pressure to give 6.567 g of a residue having >80%CBG.

A. Synthesis of Methylmagnesium Carbonate (MMC)

Methylmagnesium Carbonate (MMC) was synthesized following the protocoldisclosed by Balasubrahmanyam et al., (1973), Organic Synthesis,Collective Volume V, John Wiley & Sons, Inc., p. 439-444.

A dry 2 liter, three necked flask was fitted with a mechanical stirrer,a condenser, and a 1 liter, pressure-equalizing addition funnel, the topof which was fitted with a gas inlet tube. A clean, dry magnesium ribbon(40.0 g, 1.65 mol) was placed in the flask and the system was flushedwith nitrogen prior to the addition of anhydrous methanol (600 mL). Theevolution of hydrogen gas was controlled by cooling the reaction mixtureexternally. When hydrogen evolution had ceased, a slow stream ofnitrogen was passed through the system and the condenser was replaced bya total condensation-partial take-off distillation head. The nitrogenflow was stopped and the bulk of the methanol distilled from thesolution under reduced pressure. Distillation was stopped when stirringof the pasty suspension of magnesium methoxide was no longer practical.The system was again flushed using nitrogen and the outlet from thedistillation head was attached to a small trap containing mineral oil sothat the volume of gas escaping from the reaction system could beestimated.

Anhydrous dimethylformamide (DMF)(700 mL) was added to the reactionflask, and the resulting suspension was stirred vigorously while astream of anhydrous carbon dioxide was passed into the reaction vesselthrough the gas inlet tube attached to the addition funnel. Thedissolution of carbon dioxide was accompanied by an exothermic reactionwith the suspended magnesium methoxide. When no more CO₂ is absorbed,the colorless solution was heated under a slow stream of CO₂ gas untilthe temperature of the liquid distilling reached 140° C., indicatingthat residual methanol had been removed from the reaction mixture. Thereaction mixture was flushed using a slow stream of nitrogen to aid incooling the mixture to room temperature under an inert atmosphere. Thisyielded a solution having 536 mg MMC/mL of DMF.⁸

B. Synthesis of CBGA

6-carboxylicacid-2-[(2E)-3,7-dimethylocta-2,6-dienyl]-5-pentyl-benzene-1,3-diol,Cannabigerolic Acid (CBGA) was prepared as follows. To a 10 mL conicalflask was added 1 mL of a DMF solution of MMC. To this solution wasadded 2-[(2E)-3,7-dimethylocta-2,6-dienyl]-5-pentyl-benzene-1,3-diol(120 mg, 0.379 mmol). The flask was heated at 120° C. for 1 hour,following which the reaction mixture was dissolved in 100 mL ofchloroform:methanol (2:1) solution. The pH of this solution was adjustedwith dilute HCl to pH 2.0, and then partitioned using 50 mL H₂O.

The organic layer was dried over sodium sulfate and the solvent wasremoved by evaporation. HPLC analysis of the crude reaction showed ˜40%conversion of CBG to CBGA.

Alternatively, 3.16 g (10 mmols) of CBG (or any other neutralcannabinoid), 8.63 g (100 mmols) magnesium methylate and 44 g (1 mol) ofdry ice were sealed in a pressure compatible vessel. The vessel isheated to 50° C., and the temperature held at this value for threehours. Following heating, the vessel is cooled to room temperature andslowly vented. The reaction mixture was dissolved in 100 mL of achloroform:methanol (2:1) solvent. The pH of this solution was adjustedwith dilute HCl to pH 2.0 and this solution was then partitioned using50 mL of H₂O. The organic layer was dried over sodium sulfate and thesolvent was removed by evaporation. HPLC analysis of crude reactionmixture showed ˜85% conversion of CBG to CBGA using this protocol.

Crude CBGA was purified by chromatography using a 2.0 cm×25 cm silicagel column. The product was eluted using a mixture of n-hexane:ethylacetate (2:1) (1000 mL), to obtain 45 mg (0.125 mmol)(37.5%) of thedesired product.

Alternatively, ultra high purity CBGA was obtained by chromatographingthe crude using LH-20 lipophilic resin as the medium. 400 g of LH-20Sephadex resin was first swollen using 2 L of DCM:chloroform (4:1)solvent. The swollen resin was gravity packed in a 44×2.75 cm column.The column was loaded with 2.1 g of crude CBGA dissolved in a minimumamount of DCM:chloroform (4:1) solvent and eluted with 1.7 L of the samesolvent. 100 mL fractions were collected. The unreacted CBG was elutedas a yellow/orange solution using this solvent system. After the passageof about 1.7 L of this solvent, no more yellow/orange fraction wereobserved and the eluting solvent was changed to 100% acetone to elutethe bound CBGA.

The fractions containing CBGA were pooled and the solvent was removed toobtain 0.52 g CBGA (˜90% recovery). Increasing the volume ofDCM:chloroform (4:1) solvent passed through the column prior to elutingwith acetone, yielded CBGA having purity greater than 99.5%.

C. Synthesis of CBGV

CBGV was synthesized as follows.

A. Methyl 6-N-Propyl-2-hydroxy-4-oxo-cyclohex-2-ene-1-carboxylate

Briefly, 3-hepten-2-one (30.1 g, 0.25 mol) was added dropwise to a drymethanolic (125 mL dry MeOH), solution of diethyl malonate (52.016 g,0.323 mol) and sodium methoxide (16.206 g, 0.3 mol). The crude productweighed 46.315 g upon drying at 45° C. overnight in a vacuum oven. Thecrude product was dissolved in petroleum ether (300 mL). After stirring,any undissolved material was filtered from the solution prior to theaddition of ethyl acetate (30 mL), to precipitate CBGV. The precipitatewas filtered and dried overnight at 44° C. in a vacuum oven. A total of33.569 g (0.157 mol) (52.3%) of the desired product was recovered.

B. 1-N-Propyl-3, 5-dihydroxybenzene

A procedure similar to the one described above for the synthesis ofolivetol was used to manufacture the titled compound, except that methyl6-N-propyl-2-hydroxy-4-oxo-cyclohex-2-ene-1-carboxylate was used as thestarting material. Briefly, to a stirring ice cold DMF solution ofmethyl 6-N-propyl-2-hydroxy-4-oxo-cyclohex-2-ene-1-carboxylate was addeda DMF solution of bromine. Following the addition of bromine thereaction mixture was heated to 80° C. Heating was accompanied by thegeneration and release of carbon dioxide gas. After gas evolution hasceased, the temperature of the reaction was increased to 160° C. andheating was continued for 10 hours. The reaction was then cooled and DMFwas removed under reduced pressure. The crude mixture was diluted withwater and subjected to solvent extraction using diethyl ether. Thetitled compound was obtained by removing the ether and distilling theoil that remains.

C. 2-[(2E)-3,7-dimethylocta-2,6-dienyl]-5-propyl-benzene-1,3-diol,(CBGV)

The synthesis of CBGV proceeded by adding p-toluenesulfonic acid to achloroform solution of geraniol and 1-N-Propyl-3,5-dihydroxybenzene.After stirring the reaction at room temperature in the dark for 12hours, water was added to partition the crude product into thechloroform layer. The chloroform layer was then washed with saturatedsodium bicarbonate, dried and the organic solvent removed prior topurification as described above for the synthesis of CBG.

D. 6-carboxylicacid-2-[(2E)-3,7-dimethylocta-2,6-dienyl]-5-propyl-benzene-1,3-diol(CBGVA)

6-carboxylicacid-2-[(2E)-3,7-dimethylocta-2,6-dienyl]-5-propyl-benzene-1,3-diol,cannabigerolic Acid (CBGVA) was prepared as follows. Methyl magnesiumcarbonate (MMC) was prepared as described above. To a DMF solution ofMMC in a flask was added2-[(2E)-3,7-dimethylocta-2,6-dienyl]-5-propyl-benzene-1,3-diol. Theflask was heated at 120° C. for 1 hour, following which the reactionmixture was dissolved in a 2:1 mixture of chloroform:methanol. The pH ofthis solution was adjusted with dilute HCl to pH 2.0, and the reactionmixture was extracted using H₂O. The organic layer was dried over sodiumsulfate and the solvent was removed by evaporation.

G. Large Scale Enzymatic Production of Cannabinoids

100 ml of a 10 mM sodium phosphate buffer (pH 5.0) were placed in aglass reaction vessel equipped with oxygen gas sparger and a stirrer. Tothis solution 35 g/l of either 2-hydroxypropyl-3-cyclodextrin (HPβCD;Kleptose® HPB), a sulfobutylether β-cyclodextrin sodium salt (SBEPβCD;Captisol®), or a randomly methylated β-cyclodextrin (RMβCD) were added.The CD was added in small 5 g portions to ensure full dissolution.

2.5 g of a cannabinoid acid synthase substrate, for example, CBGA orCBGV-A or a Formula I, II or V compound, were added to the bufferedcyclodextrin solution. The molar ratio of CD to substrate was about 4:1.60 mg of purified synthase were added to the solution and the reactionmixture was incubated at 30° C. for 8 hours. Progress of the reactionwas periodically monitored by HPLC, and using an enzymatic assay todetect and quantify the evolution of hydrogen peroxide.

After 8 hours, greater than 90% of a CBGA substrate was converted toTHCA and CBCA. The ratio of THCA to CBCA was approximately 10:1 at anacidic pH of 5.0. The ratio of the CBC isomers was 5:1.

The aqueous solution was diluted 10:1 with 95% EtOH. This causescyclodextrin to precipitate out leaving the cannabinoids in solution.The cyclodextrin was vacuum filtered, washed with 1 L of 90% EtOH, anddried to permit its reuse in a future reaction. Concentration of theethanolic solution containing the cannabinoids followed suspension ofthe residue in DCM:chlorofrom (4:1) solvent yields ˜25 g crudeorange-yellow residue.

H. Large Scale Purification of Cannabinoids

Purification of cannabinoids synthesized using a method of thistechnology was accomplished chromatographically using LH-20 lipophilicresin. Briefly, 4000 g of the resin was swollen using 20 L ofDCM:chloroform (4:1). The swollen resin was gravity packed in a 44×27.5cm column. The volume of the swollen resin is ˜1350 mL. The column wasloaded with 25 g crude residue dissolved in a minimum amount of thesolvent and then washed with 4 L DCM:chloroform (4:1) solvent to eluteCBG. No cannabinoid acids were eluted from the column during thiselution.

Gradient elution with a 1:1 to 0:1 DCM:acetone solvent was used to elutethe cannabinoid acids. Each step of the gradient used one column volume(4 L) of solvent. CBCA eluted first, followed by CBGA, and then THCA.The purity of each cannabinoid was >99.5%.

The pure cannabinoids can further be processed to their neutral or“active” form by heating the acid forms at 90° C. under vacuum.Decarboxylation was quantitative to give the neutral cannabinoid. Ifnecessary, recrystallization can be performed to obtain pharmaceuticalgrade cannabinoids.

Those of skill in the art will recognize that numerous modifications andchanges may be made to the exemplary designs and embodiments describedherein and that the invention is not limited to such embodiments.

What is claimed is:
 1. A system for producing cannabinoids wherein thesystem comprises: (i) a bioreactor comprising cells stably transformedwith one or more cannabinoid genes; (ii) a reaction mixture comprising acompound according to Formula I:

wherein R is selected from —OH, halogen, —SH, or a —NR_(a)R_(b) group;R₁ and R₂ are each independently selected from the group consisting of—H, —C(O)R_(a), —OR_(a), an optionally substituted C₁-C₁₀ linear orbranched alkylene, an optionally substituted C₂-C₁₀ linear or branchedalkenylene, an optionally substituted C₂-C₁₀ linear or branchedalkynylene, an optionally substituted C₃-C₁₀ aryl, an optionallysubstituted C₃-C₁₀ cycloalkyl, (C₃-C₁₀)aryl-(C₁-C₁₀)alkylene,(C₃-C₁₀)aryl-(C₂-C₁₀)alkenylene, and (C₃-C₁₀)aryl-(C₁-C₁₀)alkynylene, orR₁ and R₂ together with the carbon atoms to which they are bonded form aC₅-C₁₀ cyclic ring; R₃ is selected from the group consisting of H,—C(O)R_(a) and C₁-C₁₀ linear or branched alkyl; and R_(a) and R_(b) areeach independently —H, —OH, —SH, —NH₂, (C₁-C₁₀) linear or branchedalkyl, or a C₃-C₁₀ cycloalkyl; and (iii) a controller configured tomodify one or more reaction conditions to modulate the ratio of thecannabinoid products, wherein the condition to be modified is pH orsolvent.
 2. The system of claim 1, wherein the cells are selected fromthe group consisting of fungal, animal, algal, and bacterial.
 3. Thesystem of claim 2, wherein the fungal cells are yeast cells.
 4. Thesystem of claim 1, wherein the cannabinoid biosynthetic gene is thetetrahydrocannabinolic acid (THCA) synthase gene.
 5. The system of claim1, wherein the cannabinoid biosynthetic gene is the cannabidiolic acid(CBDA) synthase gene.
 6. The system of claim 1, wherein cannabigerolicacid (CBGA) is a reagent in the reaction mixture.
 7. The system of claim1, wherein R is —OH and R₁ is —H or —COOH.
 8. The system of claim 1,wherein R₂ is a C₁-C₁₀ linear or branched alkyl wherein said C₁-C₁₀linear or branched alkyl is selected from the group consisting of CH₃,C₂H₅, C₃H₇, C₄H₉, C₅H₁₁, C₆H₁₃, C₇H₁₅, C₈H₁₇, iso-propyl, sec-butyl,iso-butyl, neopentyl, 2-methyl hexyl, and 2,3-dimethyl hexyl.
 9. Thesystem of claim 1, wherein R₂ is selected from the group consisting ofCH₃, C₂H₅, C₃H₇, C₄H₉, C₆H₁₃, C₇H₁₅ and C₈H₁₇.
 10. The system of claim1, wherein R₂ is C₃H₇.
 11. The system of claim 1, wherein R₂ is C₅H₁₀.12. The system of claim 1, wherein; R is —OH R₁ is —H or —COOH; R₂ is aC₁-C₁₀ linear or branched alkyl wherein said C₁-C₁₀ linear or branchedalkyl is selected from the group consisting of CH₃, C₂H₅, C₃H₇, C₄H₉,C₆H₁₃, C₇H₁₅, C₈H₁₇, iso-propyl, sec-butyl, iso-butyl, neopentyl,2-methyl hexyl, and 2,3-dimethyl hexyl; R₃ is H.